Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

Abstract

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat-fed mice. Maximum O(2) consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Delta psi(M)) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Delta psi(M) and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O(2) consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O(2) consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O(2) consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O(2) consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

Full text

OBESITY | VOLUME 18 NUMBER 8 | AUGUST 2010 1493
nature publishing group articles
adipocyte Biology
INTRODUCTION
e main fate of the fuels, glucose, and fatty acids (FAs), in
adipocytes is utilization for synthesis of triglycerides (TGs).
However, the mechanism responsible for low rates of parti-
tioning toward oxidation is not known. e ability to regulate
FA partitioning away from storage toward oxidation could be
valuable in controlling obesity. Our studies are directed at elu-
cidating control of FA oxidation. We expect that understanding
FA partitioning could lead to identication of a direct cause of
obesity and putative targets to promote its prevention.
e fate of FA in cells is mainly regulated by three enzymes:
acyl-CoA synthetase, which activates FA to the cellular metab-
olite, long-chain acyl CoA (LC-CoA); carnitine palmitoyl
transferase-1, the gatekeeper of FA entry into the mitochondria
for oxidation, and acetyl CoA carboxylase; the generator of
malonyl-CoA that regulates carnitine palmitoyl transferase-1
activity. e initial step in FA metabolism toward either TG
synthesis or oxidation is activation by acyl-CoA synthetase to
form LC-CoA. It is generally accepted that the partitioning
of LC-CoA between oxidation and storage is determined by
whether carnitine palmitoyl transferase-1 is activated to favor
oxidation or inhibited by malonyl-CoA to favor synthesis (1).
Our prior studies evaluating the expression and activity of
these three enzymes of partitioning do not explain a low rate
of FA oxidation in adipocytes (2–4).
Mitochondria and oxidative phosphorylation are impor-
tant to adipocytes. As preadipocytes undergo adipogenesis
there is a 20- to 30-fold increase in mitochondrial number in
the cell and in the concentration of numerous mitochondrial
proteins (5,6) accompanied by increased O2 consumption (7).
iazolidinediones, as a consequence of inducing adipogen-
esis, also increase mitochondrial expression in mature adi-
pocytes (8). is major increase in mitochondrial content as
adipocytes mature supports an important role for mitochon-
dria in adipocyte function. However, this role has not been well
dened, O2 consumption in adipocytes has been measured and
basal O2 consumption is similar to most other cells, increasing
with dierentiation (7) and decreasing with age (9,10) when
Respiration in Adipocytes is Inhibited
by Reactive Oxygen Species
Tong Wang1, Yaguang Si1, Orian S. Shirihai1, Huiqing Si1, Vera Schultz1, Richard F. Corkey1,
Liping Hu1, Jude T. Deeney1, Wen Guo1 and Barbara E. Corkey1
It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more
burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria,
and fat-fed mice. Maximum O2 consumption, in vitro, was determined with a chemical uncoupler of oxidative
phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/
adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal
microscopy with MitoTracker Green and their membrane potential (ΔΨM) measured using tetramethylrhodamine ethyl
ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was
assessed in mice. Addition of FCCP collapsed ΔΨM and decreased the ATP/ADP ratio. However, we demonstrated
the same rate of adipocyte O2 consumption in the absence or presence of fuels and FCCP. Respiration was only
stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations
had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of
mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O2 consumption and decreased
body fat in vivo. These studies suggest for the first time that adipocyte O2 consumption may be inhibited by ROS,
because pyruvate and NAC stimulated respiration. ROS inhibition of O2 consumption may explain the difficulty
to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by
decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.
Obesity (2010) 18, 1493–1502. doi:10.1038/oby.2009.456
The first two authors contributed equally to this work.
1Obesity Research Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.
Correspondence: Barbara E. Corkey (bcorkey@bu.edu)
Received 14 November 2008; accepted 11 November 2009; published online 24 December 2009. doi:10.1038/oby.2009.456

1494 VOLUME 18 NUMBER 8 | AUGUST 2010 | www.obesityjournal.org
articles
adipocyte Biology
expressed per milligram protein. On the other hand, despite
the low rate of FA oxidation, mitochondrial dysfunction leads
to TG accumulation (11) implying that FA oxidation is essen-
tial to a lean phenotype.
Here we show a persistent basal rate of adipocyte O2 con-
sumption despite the stimulation with the chemical uncoupler
of oxidative phosphorylation, carbonylcyanide p-triuor-
omethoxyphenylhydrazone (FCCP). We demonstrated that
stimulation of uncoupled O2 consumption required scavenging
reactive oxygen species (ROS) by pyruvate or N-acetylcysteine
(NAC). ese data suggested that ROS-inhibited O2 consump-
tion may explain the diculty in identifying eective strategies
to increase fat burning and subsequent fat loss in adipocytes.
METHODS AND PROCEDURES
In vivo studies
C57BL/6J mice were fed a high-fat diet (35% fat-derived energy from
Harlan Teklad-TD.94059). NAC was added to drinking water (3 mg/ ml)
and replaced daily except Saturday and Sunday. On day 1 and day 10 aer
the start of treatment, whole body fat mass and lean mass were measured
in v ivo without anesthesia, using EchoMRI-700/100 whole body compo-
sition analyzer (Echo Medical Systems, Houston, TX). Total body weight
was measured by direct weighing. Percentage of fat mass was calculated
as the ratio between fat mass and total body weight multiplied by 100.
Between day 12 and day 15, each mouse was placed in a single meta-
bolic cage of the VO2/VCO2 OxyMax system (Columbus Instrument,
Columbus, OH), with free access to drinking water. Food was provided
ad libitum for the rst 24 h and removed for the next 24 h. Measuring the
changes of O2 and CO2 between input and output air continuously mon-
itored respiration. Data acquired during the rst 2 h of each experimen-
tal setup were discarded to minimize random uctuation due to manual
disturbance. e mean O2 consumption and CO2 generation rates were
averaged separately in the dark period (19:00–7:00 h) and light period
(7:00–19:00 h), each under fed and fasting conditions.
Isolation of adipocytes
White adipocytes were isolated from male Sprague–Dawley rat (230–
260 g, Charles River Laboratories, Wilmington, MA) epididymal and
perirenal fat depots by collagenase digestion, as described previously
(3,12). Briey, fat pads from the perirenal and epididymal depots were
removed, and transferred into Krebs-Ringer Phosphate HEPES (KRP)
buer (containing 2% bovine serum albumin (BSA), 130 mmol/l NaCl,
4.7 mmol/l KCl, 1.24 mmol/l MgSO
4
, 2.5 mmol/l CaCl
2
, 10 mmol/l
HEPES, 2.5 mmol/l NaH
2
PO
4
, 5 mmol/l -glucose, and 200 nmol/l
adenosine, pH 7.4) at 37 °C. Adipose tissue pieces were minced, and
digested with collagenase B (1 mg/ml) in KRP buer for 35 min at 37 °C
in a shaking water bath. e fat cell suspension thus obtained was l-
tered through a 250 µm nylon mesh, and centrifuged for 15 s at 1,000
rpm. e adipocytes collected from the top phase were washed with
KRP buer three times. e cells were resuspended in three volumes
of KRP buer, allowed to equilibrate for 15 min at 37 °C, and then used
directly for the subsequent experiments. e Institutional Animal Care
and Use Committee of Boston University School of Medicine approved
the animal use protocol.
O2 consumption in vitro
O
2
consumption was measured as described previously (13–15).
Reactions were carried out in KRP buer for adipocyte O
2
con-
sumption or in standard reaction medium (containing 1% BSA,
250 mmol/l sucrose, 10 mmol/l KCl, 5 mmol/l KH
2
PO
4
, 20 mmol/l
HEPES, 2.5 mmol/l MgCl2, 0.2 mmol/l EDTA (K+ salt), pH 7.2) for
mitochondrial O
2
consumption. O
2
consumption was measured at
37 °C for adipocytes or at 25 °C for mitochondria using a Clark-type
O
2
-sensitive electrode with amplier in a stirred, water-jacketed,
closed, silicon-coated chamber as described previously (13).
Correction was made for electrode dri. e low rate of O
2
con-
sumption, observed in the presence of the medium alone and due to
O2 use by the electrode, was subtracted from the subsequent rates.
Further increases in O2 consumption were observed upon sequential
addition of cells or mitochondria. Aer recording the basal O2 con-
sumption (cell or mitochondria), fuel substrates and other chemi-
cals (FCCP, rotenone, and NAC) were added by injection through
an injection port while continuously monitoring O
2
consumption.
Results were converted into nmol of O2 used based on the dissolved
O2 in the medium of 225 µmol/l. O2 consumption by adipocytes was
expressed per milligram cell protein for ease of comparison with
other cell types. Absolute O2 consumption rates are expressed per
milligram cell protein for ease of comparison with other cell types.
In our preparations 1 mg adipocyte protein was equivalent to 2.6 ×
106 cells or 16 µg DNA.
Measurement of glucose and endogenous
FA conversion to CO2
Glucose and endogenous FA oxidation were determined by collecting
CO2 released from glucose or FA oxidation. Adipocytes were incubated
with [U-14C] glucose for glucose oxidation studies or prelabeled with
[U-
14
C] palmitate for endogenous FA oxidation. e
14
CO
2
released
from endogenous palmitate in adipocytes was determined as previ-
ously described (3,14). Briey, isolated adipocytes were prelabeled
with 1 µCi/ml [U-14C] palmitate in KRP buer (2% BSA) for 30 min at
37 °C with gentle shaking. e cells were washed with KRP buer three
times to remove labeled palmitate from the medium. Glucose conver-
sion to CO2 was done in the same media except that [U-14C] glucose
(0.027 µCi/ml) was present during the nal incubation. e incubation
was carried out in 25-cm2 culture asks sealed with a rubber stopper
from which a plastic well was suspended. Four milliliters of adipocyte
suspension (1 ml of packed cell volume) either prelabeled with [U-14C]
palmitate or containing [U-14C] glucose were incubated in the sealed
ask for 2 h at 37 °C with gentle shaking. A folded lter paper (1 cm2)
was placed inside the well. At the end of the incubation period, the 14CO2
produced by the adipocytes was released from the media by injection
of 0.5 ml of 10 N H2SO4 into the ask. β-Phenethylamine (0.3 ml) was
injected into the center well to absorb 14CO2. Aer overnight equilibra-
tion at room temperature, each center well with lter paper containing
the absorbed 14CO2 was transferred into a 20-ml scintillation vial with
5 ml of scintillation uid for β-counting. Counts from cell-free media
were used as a blank. Calculations of endogenous FA oxidation were
based on the specic activity of the prelabeled lipid pool. Calculations
of glucose oxidation were based on the specic activity of glucose in
the incubation media.
Adipocyte immobilization
e Matrigel method of Lynch et al. (16–18) was used to allow cul-
turing and microscopy of primary adipocytes for several days. ese
cells maintained their normal spherical conguration and possessed
a single large lipid droplet. Buer was removed from fat cells and
cells were washed twice and collected in a 15 ml tube. Tissue cul-
ture media (Dulbecco’s modied Eagle medium from Gibco (Grand
Island, NY) with 10% fetal bovine serum) was added to cells. Cells
were allowed to rest at 37 °C for 30 min. In the modied method, 2%
LMP Agarose (cat. no. 15517-022; Invitrogen, Carlsbad, CA) was pre-
pared with phosphate buered saline (Gibco cat. no. 14190-144) and
maintained at 37–40 °C to prevent solidication. Cells (300 µl) were
transferred to a 1.5 ml tube and mixed gently and thoroughly with
100 µl gel at 37 °C. A drop of the mixture was placed in the middle
of a culture plate (part no. P35GC-0-10-C; MatTek, Ashland, MA),
turned up side down, to allow fat cells to rise to the bottom of the
plate, and maintained at room temperature for 5 min to allow gel for-
mation. en dishes were turned right side up and culture medium
was added and cells maintained in a 37 °C incubator equilibrated
with 5% CO2.

OBESITY | VOLUME 18 NUMBER 8 | AUGUST 2010 1495
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Confocal microscopy
Mitochondria were labelled using the mitochondria-specic dyes.
MitoTracker Green and tetramethylrhodamine ethyl ester perchlorate
(TMRE, 7 nmol/l) were from Molecular Probes, (Eugene, OR). Freshly
prepared TMRE was added to culture in ethanol from a 1,000,000 ×
stock to give a nal concentration of 7 nmol/l and incubated for 1 h
prior to visualization. Confocal microscopy was performed on live cells
using a Leica Confocal microscope (TCS SP2) with the following lasers
Kr (568/20 mW), and Ar (488/20 mW).
Mitochondrial membrane potential (ΔΨM)
To image ΔΨM in fat cells labelled with TMRE, cells were excited with
a 568-nm laser, and emission was recorded through a BP 650–710-nm
l te r. Z sections of 1,024 × 1,024 images were obtained using a ×100
immersion oil lens. In this way both perinuclear and peripheral mito-
chondria were imaged. Metamorph image analysis soware was used
for image processing and analysis. As described by O’Reilly et al. (19),
the ΔΨM-dependent component of TMRE accumulates in a Nernstian
fashion that can be described by the intensity of its uorescence. e
non-ΔΨM-dependent component of TMRE, also known as the bind-
ing component, was ignored as it is xed and voltage-independent.
For microplate assay, cells were incubated with 20 nmol/l TMRE in the
loading buer (containing 140 mmol/l NaCl, 6 mmol/l KCl, 1 mmol/l
MgCl2, 5 mmol/l HEPES, 1.8 mmol/l CaCl2, 5.8 mmol/l D-glucose, pH
7.4) for 30 min at 37 °C. en cells were washed three times and incu-
bated with KRP buer (1% BSA). Basal uorescent intensity (Ex.549/
Em.574) was measured by a temperature-controlled microplate reader
(Sare2; Tecan US, Durham, NC) at 37 °C. Next, cells were treated with
FCCP (30 μmol/l), NAC (10 mmol/l), palmitate (0.4 mmol/l) or buer,
and the kinetic uorescent intensities were recorded immediately for
10 min. For data analysis, the uorescent intensities aer chemical addi-
tion were rst normalized to their respective basal values, then compar-
ing with the appropriate control group at same time point. Final results
were calculated by averaging the ratios between treated and control
group at all time points.
ATP/ADP assay
Cells were extracted in cold 1% (wt/vol) trichloroacetic acid. Fat was
removed by washing with two volumes of chloroform. Trichloroacetic
acid was removed from the aqueous layer with four equal volume washes
of diethyl ether using vacuum suction to completely remove the ether
between each wash. Neutralized supernatants were then freeze-dried
in a SpeedVac (Savant, ermo Scientic, Waltham, MA) and stored
at −80 °C until assayed. Dried samples were redissolved in water and
an aliquot removed to measure adenosine triphosphate (ATP) directly
with luciferase using a Turner Model 20e luminometer equipped with a
Cavro injector. Adenosine diphosphate (ADP) was converted to ATP to
be assayed by luciferase, aer the sample was depleted of endogenous
ATP with ATP sulphurylase (20). Data are presented as the ATP/ADP
ratio, making them independent of changes in cell number per well or
loss of sample volume during extraction (20).
Adipocyte and liver mitochondria
Mitochondria from adipocytes and liver were prepared according to
standard procedures (21) and as described previously (4,22) with a slight
modication. Adipocytes prepared from epididymal and perirenal fat
pads or minced liver were suspended in two volumes of ice-cold homog-
enization buer (containing 0.25 mol/l sucrose, 20 mmol/l HEPES,
0.2 mmol/l EDTA (K+ salt) pH 7.4), and homogenized in a loose tting
homogenizer. Homogenates were centrifuged at 600 g for 10 min at 4 °C.
e supernatant was centrifuged at 10,000 g for 10 min at 4 °C. e pellets
containing mitochondria were suspended in 10 volumes of 0.25 mol/l
sucrose and centrifuged at 10,000 g for 10 min at 4 °C. e mitochon-
drial pellet obtained was resuspended in homogenization buer to give
a nal concentration of 1–3 mg of mitochondrial protein/ml, and stored
on ice. Protein concentration of adipocyte and liver mitochondria was
determined with the Bradford reagent using BSA as a standard.
ROS measurement
Intracellular ROS levels were measured using the 5-(and-6)-
chloromethyl-2′, 7′-dichlorodihydrouorescein diacetate, acetyl ester
(CM-H
2
DCF-DA, Molecular Probes), a cell permeable nonuores-
cent precursor. is dye measures H2O2, ROO−, and ONOO−. Within
the cells, CM-H2DCFH-DA is hydrolyzed by nonspecic esterases to
release CM-H
2
DCF, which is readily oxidized by intracellular ROS.
e oxidized product emits green uorescence (Ex.475/Em.515). e
protocol was modied from manufacture’s instruction. Briey, cells
were loaded with 0.5 μmol/l CM-H
2
DCF-DA for 20 min at 37 °C in
the same loading buer used in TMRE microplate assays. en cells
were washed three times and incubated with KRP buer (1% BSA)
for 1 h at 37 °C, with or without NAC (10 or 20 mmol/l), pyruvate
(5 mmol/l), or H2O2 (1 mmol/l). e uorescence intensity was meas-
ured by a microplate reader.
Materials
Collagenase B was from Boehringer Mannhein (Mannhein, Germany),
LMP from Invitrogen, TMRE, CM-H2DCF-DA from Molecular Probes,
Luciferase from Becton Dickinson (Bedford, MA), BSA FA-free from
American Bioanalytical (Natick, MA), media and sera from Gibco,
dishes from MatTek. Glass bottom culture dishes were from MatTek.
Matrigel Matrix was from Becton Dickinson. Other chemicals were
from Sigma Chemical (St Louis, MO). [U-
14
C] glucose and [U-
14
C]
palmitate were from NEN (Boston, MA).
Statistical analysis
Microcal Origin 50 (Microcal Soware, Northampton, MA) was used
for statistical analysis. Values are shown in gures and tables as means
of the number of separate measurements (n) ± s.e. Comparisons were
performed using ANOVA. P < 0.05 was considered signicant.
RESULTS
Influence of fuels on adipocyte O2 consumption
Cellular O2 consumption is mainly a reection of mitochon-
drial respiration from glucose and FA to generate a constant
level of ATP and maintain a highly negative mitochondrial
membrane potential to drive ATP production. To study con-
trol of adipocyte respiration, fuel-stimulated O2 consumption
in intact cells was evaluated. Table 1 shows that basal (5 mmol/l
glucose) adipocyte O2 consumption (4.3 ± 0.18 nmol/min/
mg protein, n = 65) is within the range of values reported for
other cell types (23–25) when expressed per milligram protein.
Basal O2 consumption is normally about 25–30% of maximal
O2 consumption. However, unlike other cells, FCCP addition
did not increase O
2
consumption in adipocytes (Ta b l e 1 , top
row, FCCP column). e possibility that insucient substrate
supply could account for the failure to increase uncoupled O2
consumption appeared to be unlikely because addition of a
wide variety of substrates including acetate, octanoate, methyl-
succinate, malate, glutamine, lactate, and combinations thereof
caused only small but insignicant changes in O2 consumption
(Table 1).
Interaction of FA and oxidative metabolism
e possibility that the high-fat environment of adipocytes
might inhibit fuel oxidation was considered because the meta-
bolically active intracellular FA product, LC-CoA, has been
reported to inhibit O2 consumption and substrate transport
into the mitochondria (26–28). However, exogenous FA did
not inhibit either glucose or endogenous FA oxidation in

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adipocyte Biology
adipocytes but rather exerted a stimulatory eect on glucose
oxidation at high FA concentrations (Supplementary Figure
S1a online). Consistent with the lack of eect of FA on endog-
enous FA oxidation (Supplementary Figure S1b online),
exogenous FA did not signicantly stimulate O2 consumption
either (2.66 ± 0.26 and 3.42 ± 0.37 nmol/min/mg, control vs.
0.5 mmol/l palmitate, n = 9). We previously documented lit-
tle eect of glucose on FA oxidation (4). us, although FA
oxidation is very low in adipocytes, FA do not appear to inhibit
either glucose or FA oxidation under the conditions tested.
The pyruvate effect
In contrast to the lack of FCCP stimulation of respiration by
other fuels and fuel combinations, pyruvate stimulated more
than a threefold increase in O2 consumption in uncoupled
adipocytes (Table 1, bottom row, and Figure 1a). Stimulation
of uncoupled O2 consumption by pyruvate was concentra-
tion-dependent with a half-maximal eect at ~0.3 mmol/l
(Figure 1a). Stimulated O
2
consumption was also dependent
on the concentration of FCCP with a rather high maximal
eect at about 20 µmol/l (Figure 1b). is high concentration
of FCCP may be due to its lipophilicity and distribution into
the fat droplet. Pyruvate is metabolized in the cytosol where it
can be converted to lactate via lactate dehydrogenase and in
the mitochondria where it serves as a source of acetyl CoA for
the citric acid cycle.
To determine whether pyruvate action occurred in mito-
chondria or cytosol, we tested the ability of the mitochondrial
pyruvate transport inhibitor α-cyanocinnamic acid (Cyncin)
(29), to modulate pyruvate stimulation of FCCP-induced O2
consumption. Figure 1c shows that Cyncin inhibited basal
O2 consumption suggesting that a major component of basal
respiration required pyruvate transport into the mitochon-
dria. Cyncin also markedly abrogated the stimulatory eect
of pyruvate plus FCCP on O2 consumption to levels below
basal. In order to rule out the possibility that Cyncin nonspe-
cically inhibited the plasma membrane pyruvate carrier, we
evaluated methyl-pyruvate (MePyr), which is a carrier-inde-
pendent cell permeant ester that is converted to pyruvate in
the cytosol following methyl ester cleavage by esterases. As
shown in Figure 1c, MePyr was nearly as eective as pyru-
vate in stimulating O2 consumption with FCCP and this
eect was totally blocked by Cyncin. ese data indicate that
pyruvate must enter the mitochondria to exert its stimulatory
eect, implicating an intramitochondrial site of inhibited O
2
consumption.
Sensitivity of adipocyte mitochondria to uncoupling
Chemical uncouplers like FCCP act as proton ionophores to
collapse the mitochondrial membrane potential. is causes
a decrease in the ATP/ADP ratio due to loss of the proton
motive force needed to synthesize ATP. To determine whether
adipocyte mitochondria were initially coupled and became
uncoupled in response to FCCP, we measured mitochondrial
membrane potential (ΔΨM and the cellular ATP/ADP ratio. For
ΔΨM assessments, adipocytes were immobilized upside down
in a gel. Mitochondrial localization was determined using the
mitochondria-specic dye, MitoTracker green. Figure 2a (le)
shows a three dimensional reconstruction of an immobilized
spherical adipocyte with a high concentration of mitochondria
in the vicinity of the nucleus. Figure 2a also illustrates a sin-
gle plane through an immobilized adipocyte before (middle)
and aer (right) FCCP addition. Comparing the intensity of
the TMRE images as well as the merged images, it is clear that
FCCP addition decreased ΔΨM. is result was also conrmed
using a microplate assay (Figure 2b). NAC is a powerful anti-
oxidant that also increases cellular glutathione content, thus
facilitating removal of H2O2 by glutathione peroxidase (30).
We found that NAC decreased ΔΨM. Palmitate also decreased
ΔΨM though less extensively.
To determine whether the decreased ΔΨM had the expected
eect of lowering the ATP/ADP ratio, adipocytes were incu-
bated with and without FCCP for 10 min and the ratio deter-
mined in deproteinized cell extracts. Figure 2c shows that the
basal ATP/ADP ratio was high (12.1 ± 1.4) as expected with
well-coupled adipocyte mitochondria. ere was little eect
of pyruvate addition on the ratio, consistent with an adequate
endogenous supply of mitochondrial substrate. As expected,
FCCP reduced the ATP/ADP ratio to <1 (Figure 2c).
Taken together, the data from Figures 2a–c indicate that
FCCP eectively uncoupled previously well-coupled adipocyte
mitochondria, a maneuver that generally elicits maximum O2
consumption as cells strive to restore the mitochondrial mem-
brane potential and ATP/ADP ratio using any available fuel.
However, uncoupling did not stimulate O2 consumption in
adipocytes in the absence of pyruvate as illustrated in Table 1
and Figure 1c.
Table 1 O2 consumption of rat white adipocytes with different
substrates
Substrate
n
O2 consumption
(nmol/min/mg) n
O2 consumption
(nmol/min/mg)
Control FCCP
Basal (5 mmol/l
Glucose)
65 4.25 ± 0.18 12 3.80 ± 0.43
Methyl-succinate 4 3.37 ± 0.47 4 5.53 ± 1.34
Methyl-succinate/
octanoate
3 4.29 ± 1.41 3 5.80 ± 1.83
Acetate 3 7.48 ± 1.95 3 5.20 ± 1.36
Glutamine 3 4.23 ± 1.37 3 4.74 ± 0.96
Octanoate 3 4.77 ± 1.53 3 8.28 ± 2.66
Glutamine/octanoate 3 4.22 ± 0.87 3 3.99 ± 0.98
Malate 3 4.37 ± 1.50 3 6.42 ± 2.30
Lactate 3 4.69 ± 1.13 3 5.35 ± 1.45
Pyruvate 10 4.48 ± 0.87 15 13.95 ± 1.55
O2 consumption in intact adipocytes was measured in 1 ml of adipocyte sus-
pension (adipocytes/KRP buffer 1:3, vol/vol), and addition of FCCP (30 µmol/l),
octanoate (1 mmol/l), methyl-succinate (5 mmol/l), glutamine (2 mmol/l), acetate
(5 mmol/l), lactate (5 mmol/l), malate (5 mmol/l), pyruvate (5 mmol/l), or combina-
tions, as indicated. Data are means ± s.e. of experiments from “n” separate
preparations.

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Isolated adipocyte mitochondria
To determine whether adipocyte mitochondria were unusual
in their fuel preferences and possibly preferred pyruvate as sub-
strate due to expression of the mitochondrial pyruvate carrier
preferentially over other anion carriers, the fuel preferences
of mitochondria isolated from adipocyte and liver were com-
pared. Table 2 shows that the isolated mitochondria from both
liver and adipocytes were well coupled. e data also indicate
that succinate elicited the greatest rate of O
2
consumption in
adipocyte mitochondria as has been shown in mitochondria
from other cell types (31) and, although pyruvate was an excel-
lent fuel, succinate was better. us, an unusual preference for
pyruvate or transport of pyruvate could not explain the unique
ability of this substrate and not succinate to enhance uncoupled
O2 consumption in adipocytes. is unique preference for
pyruvate suggested that pyruvate stimulation of uncoupled O2
consumption was not a consequence of its fuel function.
Adipocyte ROS
In addition to its well-established roles as a fuel, oxidant in
the cytosol (via lactate dehydrogenase) and reducing agent
in the mitochondria (via pyruvate dehydrogenase), pyruvate
has long been known to act as a chemical scavenger of ROS
(32,33). We tested the possibility that it was ROS scaveng-
ing that explained pyruvate’s ability to stimulate uncoupled
O2 consumption. First, we found that cells exposed to NAC
20
a
c
d
b
16
12
O
2
consumption
(nmol/min/mg protein)
O
2
consumption
(nmol/min/mg protein)
O2 consumption
(nmol/min/mg protein)
O2 consumption
(nmol/min/mg protein)
8
4
0
30
25
20
15
10
5
0
0510 15 20 25 30 35 40 45
0 0.1
16
14
12
10
8
6
4
0
2
30
25
20
15
10
5
0
FCCPBasal
BasalNAC
Cyncin Pyr Pyr
FCCP
Pyr
FCCP
Cyncin
M-Pyr M-Pyr
FCCP
M-Pyr
FCCP
Cyncin
0.3
Pyruvate concentration (mmol/l) FCCP concentration (µmol/l)
0.5 1.0 5.0
*
*
*
*
**
Figure 1 O2 consumption of isolated rat white adipocytes in the presence of different compounds. (a) Concentration dependence of pyruvate
stimulated uncoupled O2 consumption. O2 consumption in intact adipocytes was measured at 37 °C in 1 ml of adipocyte suspension (adipocytes: KRP
buffer 1:3, vol/vol), with 5 mmol/l glucose and 30 µmol/l carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) plus the indicated concentrations
of pyruvate. Data are means ± s.e. of experiments from three separate preparations. (b) Concentration dependence of FCCP stimulated
O2 consumption. O2 consumption in intact adipocytes was measured as described above with 5 mmol/l pyruvate plus the indicated concentrations of
FCCP. Data are means ± s.e. of experiments from three separate preparations. (c) The influence of different substrates and inhibitors on adipocyte
O2 consumption. O2 consumption was measured under basal conditions or following addition of FCCP (30 µmol/l), Cyncin (5 mmol/l), pyruvate
(5 mmol/l), MePyr (5 mmol/l) separately, or in combinations indicated. Data are means ± s.e., n = 4. (d) N-acetylcysteine (NAC) stimulated O2
consumption. O2 consumption was measured under basal conditions or following addition of 10 mmol/l NAC. Data are means ± s.e., n = 4. *P < 0.05.

1498 VOLUME 18 NUMBER 8 | AUGUST 2010 | www.obesityjournal.org
articles
adipocyte Biology
exhibited a stimulation of O2 consumption even greater than
pyruvate, supporting the concept that ROS plays a role in the
inhibition of O2 consumption (Figure 1d). Interestingly, NAC
tended to decrease both ΔΨM (Figure 2b) and the ATP/ADP
ratio (Figure 2c), which may have contributed to its ability to
stimulate respiration. However, these changes were not statisti-
cally signicant.
Second, we assessed ROS levels directly in isolated adipocytes
using CM-H2DCF-DA. Figure 2d illustrates that the presence
of pyruvate, FCCP or both signicantly decreased ROS by
15–30% whereas NAC decreased ROS by 40% in adipocytes,
as reported previously by others (30,34,35). e addition of
H2O2, as a positive control, caused a large increase in uores-
cence. ese data are consistent with the well-established role
of pyruvate and NAC as ROS scavengers.
ird, we examined whether the role of pyruvate as ROS
scavenger and energy substrate could be distinguished in
FCCP-treated fat cells. Rotenone is a chemical inhibitor of
mitochondrial complex I. At a concentration of 1 μmol/l and
above, rotenone completely inhibited FCCP-stimulated respi-
ration in the presence of pyruvate (Figure 3a). However, fur-
ther addition of methyl-succinate, a substrate of mitochondrial
complex II that bypasses the rotenone block in the respiratory
chain, signicantly increased respiration by 129% over basal
(Figure 3b, black bars). On the other hand, methyl-succinate
only increased respiration by 34% in the absence of pyru-
vate (Figure 3b, gray bars). us, in the presence of FCCP to
uncouple, and pyruvate to scavenge ROS, methyl-succinate
stimulated respiration although it was unable to do this alone
(Table 1).
In vivo ROS scavenging
Previous studies indicated that NAC increases O2 consumption
in rats (36) and reduces visceral fat in mice (37). To assess the
possible relevance of ROS scavenging on fat mass in vivo, mice
were given NAC in their drinking water. Aer 10 days of high-
fat feeding, compared to Day 1, there was signicantly less gain
in body fat as assessed by NMR (Figure 4a) with no dierence
in body weight gain in mice given NAC. Aer 12–15 days on
the diet, the both fed and 18 h fasted mice given NAC had sig-
nicantly higher O2 consumption and CO2 production than the
controls (Figure 4b). ese dierences were most pronounced
during the light cycle or inactive period implying an increase
in resting energy expenditure.
Figure 2 Adipocyte mitochondrial membrane potential, adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, and reactive oxygen
species (ROS). (a) Left, three dimensional reconstruction of adipocyte mitochondria identified with MitoTracker Green (MTG). Single adipocytes
exhibited a high concentration of mitochondria in the vicinity of the nucleus and sparse distribution of mitochondria around the equator. Middle
and right, illustration of decreased mitochondrial membrane potential in adipocytes following carbonylcyanide p-trifluoromethoxyphenylhydrazone
(FCCP) addition. A single plane through an immobilized adipocyte before (middle) and after (right) FCCP addition. Adipocytes were loaded with
MTG to identify mitochondria (green image in upper left panels) and tetramethylrhodamine ethyl ester perchlorate (TMRE) to measure mitochondrial
ΔΨ (red image upper right panels). The merged images are shown in the lower right panels. Illustration is from one of three separate preparations.
(b) Mitochondrial membrane potential using a microplate assay. Please refer to the Methods section for data normalization. Data are means ± s.e.,
n = 3. *P < 0.05. (c) The influence of FCCP, pyruvate, N-acetylcysteine (NAC), and combinations on the ATP/ADP ratio. The ATP/ADP ratio was
determined in adipocytes following incubation with KRP buffer (adipocytes:KRP buffer 1:3, vol/vol) for 10 min in the presence of FCCP (30 µmol/l),
pyruvate (5 mmol/l), pyruvate plus FCCP, NAC (10 mmol/l), or NAC plus FCCP. Data are means ± s.e., n = 3. *P < 0.05. (d)The effect of pyruvate,
NAC, FCCP, or H2O2 on ROS levels measured using CM-H2DCF. ROS levels were measured using CM-DCF at excitation, 490 nm, emission, 529
nm. Cells were first loaded with CM-H2DCF and then incubated with pyruvate (5 mmol/l), NAC (10 or 20 mmol/l) or H2O2 (100 mmol/l) in KRP buffer
(5 mmol/l glucose, 1% BSA) as described in methods. Graphs represent experiments that were repeated 5–11 times. Data are means ± s.e. *P < 0.05.
0
Basal FCCP Pyr Pyr NAC NAC
FCCP
***
2
4
6
8
10
ATP/ADP ratio
12
14
c
−50
−40
−30
−20
−10
0
Control PYR FCCP
*
PYR
FCCP
*
NAC
*
H2O2
*
10
% Change of DCF fluorescent intensity
20
30
40
50
d
0
Palmitate
0.4 mmol/l
NAC
10 mmol/l
FCCP
30 µmol/l
b
*
% Decrease of TMRE
fluorescent intensity
−5
−10
−15
MTG TMRE TMRE
Merge
Before FCCP After FCCP
a
Merge
MTG
*

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DISCUSSION
e surprising nding from these studies is that O2 consump-
tion above basal is inhibited in adipocytes but can be stimu-
lated by removing ROS. is implies either high sensitivity of
adipocyte O2 consumption to normal levels of ROS or high
levels of ROS in adipocytes. e latter explanation is more
likely because many studies have documented that increased
adiposity is accompanied by increased oxidative stress and
inltration of adipose tissue with macrophages. is suggests
that inhibition of O2 consumption and high levels of ROS are
tolerated and possibly serve an important function in adi-
pocytes unlike other cell types where oxidative stress and ROS
lead to apoptosis. Such unique tolerance could lead to a thriy
phenotype by limiting fat burning and promoting fat storage.
ROS and mitochondrial function
It is known that various reactive species inhibit O2 consump-
tion and stimulate apoptosis (38–40). Data from obese mice
indicate ROS levels increase selectively in adipose tissue (35).
Table 2 O2 consumption of isolated mitochondria from rat white adipocytes and liver with different substrates
Substrates
O2 consumption (nmol/mg protein/min)
Adipocyte mitochondria Liver mitochondria
nState 4 State 3 State 3/State 4 nState 4 State 3 State 3/State 4
Succinate 5 84 ± 21 271 ± 79 3.30 ± 0.36 6 22 ± 20 156 ± 13 7.10 ± 0.28
l-Glutamine plus malate 4 41 ± 11 96 ± 21 2.39 ± 0.12 3 12 ± 0.70 48 ± 13 4.58 ± 0.35
Pyruvate plus malate 4 20 ± 3 159 ± 39 7.91 ± 0.58 5 15 ± 2.68 49 ± 13 3.15 ± 0.27
O2 consumption was measured continuously following sequential addition of substrate (succinate (5 mmol/l), or pyruvate (5 mmol/l) or glutamine (5 mmol/l) plus malate
(1 mmol/l) and ADP (0.2 mmol/l). State 4 O2 consumption reflects O2 consumption without and State 3, with ADP added. Data are means ± s.e. of “n” experiments from
separate preparations.
Figure 3 Effect of inhibiting pyruvate oxidation on respiration with
methyl-succinate (MeS) as an alternative substrate. Reagents shown
on the x axis were added sequentially and their concentrations
are indicated. (a) Rotenone dose-dependently inhibited uncoupled
O2 consumption driven by pyruvate. Data shown are fold-change
of O2 consumption rate over basal condition. (b) MeS stimulated
O2 consumption in the presence of rotenone and carbonylcyanide
p-trifluoromethoxyphenylhydrazone, with (black bar) or without (gray bar)
pyruvate. Data shown are relative change of O2 consumption rate from
basal respiration. Data are means ± s.e., n = 3. *P < 0.05.
Before
FCCP 30 µmol/l
PYR 5 mmol/l
0.5
1
1.5
2
Rotenone (µmol/l)
*
O
2
consumption
(Fold change over basal)
15
a
b
10
5
0
2
1
Fold change from basal respiration
3
0
No pyruvate
1
µmol/l Rotenone &
30 µmol/l FCCP
With pyruvate
5 mmol/l Pyruvate
5 mmol/l MeS
Figure 4 Effect of N-acetylcysteine (NAC) in vivo. (a) Body fat and body
weight. Postweaning male mice were fed a high-fat diet (35% fat-derived
energy). NAC (3 mg/ml) was added to their drinking water and replaced
daily (black bars). On days 1 and 10 mice were weighed and analyzed
by NMR for fat mass. (b) O2 consumption and CO2 production. Between
day 12 and day 15, mice were analyzed by indirect calorimetry for
respiratory rate and CO2 production. Data are means ± s.e. for four mice.
*P < 0.05.
30
a
b
20
Day 1
Day 1
Day 10 Day 10
15
10
5
0
25
20
Body weight (g)
Body fat
15
10
5
0
4
*
*
*
FedFasted FedFa sted
3
Mean O
2
consumption
(l/kg/h)
2
1
0
7 pm–7 am
7 am–7 pm
7 pm–7 am
7 am–2 pm
7 pm–7 am
7 am–7 pm
7 pm–7 am
7 am–2 pm
4
3
Mean CO
2
consumption
(l/kg/h)
2
1
0
Vehicle
NAC
NAC
Vehicle
NAC
Vehicle
NAC
Vehicle

1500 VOLUME 18 NUMBER 8 | AUGUST 2010 | www.obesityjournal.org
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Our data and other studies in the literature suggest that ROS
levels are high in adipocytes (30,34), suggesting unusual toler-
ance. Mitochondrial function is also important for TG storage
in adipocytes. It has been shown previously that mitochon-
drial dysfunction in adipocytes leads to increased TG storage
(11) and that aging is accompanied by decreased mitochon-
drial function and increased lipid stores (41–43). Our ndings
suggest that limited mitochondrial O2 consumption occurred
spontaneously and may be one of the unique attributes of
adipocytes.
Increased ROS or increased ROS sensitivity
Adipocytes isolated from mice fed a high-fat diet (35) or
exposed to nutrient excess in vivo (30) display signicantly
elevated ROS in vitro. us, adipocytes appear to reside in a
relatively high ROS environment due both to endogenous
and exogenous ROS production from resident macrophages.
Despite such a potentially damaging environment, adipocytes
appear relatively insensitive to the damaging eects of ROS.
Dierentiation of murine 3T3-L1 preadipocytes into adi-
pocytes is associated with the acquisition of apoptotic resist-
ance accompanied by upregulation of cell survival genes
even under conditions where ROS production is increased
(44). Interestingly, human cells appear to be protected from
apoptosis through an autocrine/paracrine action of IGF-1,
which maintains the expression of antiapoptotic proteins,
Bcl- (XL) and Fas–associated-death-domain protein like IL-1-
converting-enzyme-inhibitory protein (45). us, ROS and
inammation exist in adipocytes (30,34,35) and this appears
to occur in response to the same stimuli that increase ROS in
other cells but these do not lead to the expected increase in
damage, presumably due to the protective eect of antiapop-
totic proteins (45–47). e mechanistic basis for tolerance of
high levels of ROS requires further investigation.
ROS scavenging and pyruvate
Pyruvate has several fates in the cell. It is converted to lactate
in the cytosol with accompanying conversion of NADH to
NAD. is cannot explain our ndings because the data show
that stimulation of O2 consumption by FCCP in adipocytes
requires intramitochondrial pyruvate. Pyruvate is also con-
verted to acetyl CoA in the mitochondria with accompanying
conversion of NAD to NADH. Although this is likely to occur,
it also cannot explain our ndings because other mitochon-
drial fuels also increase mitochondrial NADH but do not stim-
ulate respiration (Ta b l e 1 ). In addition, we were still able to
take advantage of the ability of pyruvate to scavenge ROS aer
inhibiting its metabolism with rotenone and using an alterna-
tive substrate, succinate (administrated as methyl-succinate),
that bypassed the respiratory block. Our ndings are consist-
ent with other diverse ndings reported in the literature. e
ability of pharmacological levels of pyruvate to protect cells by
nonenzymatic scavenging of ROS has been demonstrated in
cardiac cells and neurons (32,33,48–49) in addition to our nd-
ings here. is is not surprising because most ROS is produced
in the mitochondria.
e reason why NAC is more powerful than pyruvate
(Figure 1c vs. 1d) may relate to its greater oxidative potential
or to its ability to slightly decrease the ΔΨ
M
(Figure 2b) and
ATP/ADP ratio (Figure 2c). Clearly the decrease in ΔΨM was
not sucient, although it may be necessary, because FA also
slightly depolarized the mitochondria but did not stimulate
respiration. NAC may also increase glutathione within the
mitochondria, which may be important if glutathione levels
are low in adipocytes. Glutathione levels in adipocyte mito-
chondria have not been reported. Alternatively, glutathione
generated by NAC may cause the mitochondrial redox state
to be more oxidized (50,51) whereas pyruvate causes a more
reduced state. It has been shown that a highly reduced state
favors ROS generation. In addition, mitochondrial eux of
H2O2 may also exert additional eects in the cytosol. ere
may also be a component of the cytosolic glutathione redox
state that contributes to respiratory inhibition.
Interestingly, the in vivo eect of NAC on respiration and
body fat has also been reported in other studies. Novelli et al.
demonstrated that in Wistar rats on a high-sucrose diet, 30-day
NAC feeding increased O2 consumption, decreased respiratory
quotient without aecting total energy intake (36). In the other
study by Kim et al., injecting C57BL/6 mice with NAC for 8 weeks
dose-dependently decreased visceral fat mass and body weight
(37). ough the authors of the second study suggested that NAC
inhibits preadipocyte dierentiation, our conclusion on NAC-
induced respiration could oer an alternative explanation.
Inhibition of O2 consumption
e documentation of uncoupling in intact cells in the pres-
ence of adequate fuel and O
2
without an increase in O
2
con-
sumption is unusual and has not been previously reported to
our knowledge. In most cells decreased mitochondrial ΔΨ
M
,
chemical uncoupling of oxidative phosphorylation or a large
fall in the ATP/ADP ratio, as we found (Figures 2b and c), stim-
ulate O
2
consumption (26–28). Among other putative factors
known to inhibit O
2
consumption, we ruled out FA-induced
inhibition of fuel oxidation (Supplementary Figure S1 online),
lack of fuel (Table 1), O2 deprivation (through direct measure-
ment) and high ATP levels in the presence of an uncoupler
(Figure 2c). Although FA and glucose have been shown to
compete for entry into the Krebs cycle in many cells, this does
not occur to a signicant extent in adipocytes (Supplementary
Figure S1 online). Because LC-CoA levels are likely to be high
in adipocytes in order to handle the high FA uxes, another
possibility is inhibition of the adenine nucleotide translocase or
other mitochondrial anion carriers (52–57) by LC-CoA. None
of these mechanisms appear to explain inhibition of uncou-
pled respiration in adipocytes since it can be overcome by ROS
removal. Furthermore, administration of NAC to whole ani-
mals increases O2 consumption and decreases body fat sug-
gesting that ROS scavenging or provision of glutathione from
NAC also stimulates respiration in vivo. It should be noted
that others have attributed NAC eectiveness to alterations
in β-hydroxyacyl CoA dehydrogenase (58) or metalothionine
II (37). ese correlations may be relevant, are not mutually

OBESITY | VOLUME 18 NUMBER 8 | AUGUST 2010 1501
articles
adipocyte Biology
exclusive but do not provide a mechanistic explanation for the
observed increase in respiration.
The thrifty phenotype
e concept of a thriy phenotype was introduced by Hales
in 1992 to explain a high incidence of type 2 diabetes in indi-
viduals exposed to gestational malnourishment (59). e
rationale for such a mechanism is to adapt the unborn child
to survival under circumstance of inadequate nutrition; how-
ever, a molecular mechanism underpinning this phenomenon
has not been elucidated. e data presented here suggest that
genes that prevent fat burning and promote storage are among
the attributes of normal fat cells. us, fat cells by virtue of
their unique function exhibit a thriy phenotype. e particu-
lar genes involved could include those that confer protection
from cell damage. Deprivation, whether gestational or due to
dieting, could induce these genes. It is well-established that the
genetic prole of a reduced obese individual is dierent from
a never-obese individual of the same weight (60,61). us, a
thriy phenotype is hypothesized to result from nutritional
excess in individuals who are unable to overcome ROS inhibi-
tion of O2 consumption in their fat cells. It will be interesting to
determine whether there are variations in ROS sensitivity that
correlate with obesity and leanness. A further implication of
this hypothesis is that excess ROS in other cell types might also
inhibit O2 consumption and cause ectopic fat storage.
The model
A testable model that evolves from our data has four impor-
tant constituents: (i) high levels of FA, as occur normally in fat
cells, increase ROS production; (ii) a relatively sluggish scav-
enging system, particularly in mitochondria, allows elevated
ROS levels to be maintained; (iii) ROS inhibits O
2
consump-
tion; (iv) an enhanced adipocyte defense system tolerates levels
of ROS sucient to inhibit O2 consumption without inducing
cell damage. As a result, fat cannot be burned and TG storage
is favored. It is predicted that preventing or reversing the four
constituents described above will decrease TG stores and test
the model. Testing this model is the focus of our current work.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at
http://www.nature.com/oby
ACKNOWLEDGMENTS
This work was supported by NIH grants: DK56690, DK46200 (B.E.C.),
DK74778 (O.S.), and DK59261 (W.G.).
DISCLOSURE
The authors declared no conflict of interest.
© 2009 The Obesity Society
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