From the Diabetes Unit, Department of Medicine,
Division of Endocrinology and Metabolism, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, Massachusetts 02215 and the
Endocrine Research Division, Lilly Research Laboratories, Eli
Lilly and Company, Indianapolis, Indiana 46285
Received for publication, December 26, 2000
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ABSTRACT |
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UCP3 is a mitochondrial membrane protein
expressed in humans selectively in skeletal muscle. To determine the
mechanisms by which UCP3 plays a role in regulating glucose metabolism,
we expressed human UCP3 in L6 myotubes by
adenovirus-mediated gene transfer and in H9C2
cardiomyoblasts by stable transfection with a tetracycline-repressible UCP3 construct. Expression of UCP3 in L6
myotubes increased 2-deoxyglucose uptake 2-fold and cell surface GLUT4
2.3-fold, thereby reaching maximally insulin-stimulated levels in
control myotubes. Wortmannin, LY 294002, or the tyrosine kinase
inhibitor genistein abolished the effect of UCP3 on glucose
uptake, and wortmannin inhibited UCP3-induced GLUT4 cell surface
recruitment. UCP3 overexpression increased phosphotyrosine-associated
phosphoinositide 3-kinase (PI3K) activity 2.2-fold compared with
control cells (p < 0.05). UCP3 overexpression
increased lactate release 1.5- to 2-fold above control cells,
indicating increased glucose metabolism. In
H9C2 cardiomyoblasts stably transfected with
UCP3 under control of a tetracycline-repressible promotor,
removal of doxycycline resulted in detectable levels of UCP3 at 12 h and 2.2-fold induction at 7 days compared with 12 h. In
parallel, glucose transport increased 1.3- and 2-fold at 12 h and
7 days, respectively, and the stimulation was inhibited by wortmannin
or genistein. p85 association with membranes was increased 5.5-fold and
phosphotyrosine-associated PI3K activity 3.8-fold. In contrast,
overexpression of UCP3 in 3T3-L1 adipocytes did not alter
glucose uptake, suggesting tissue-specific effects of human UCP3. Thus,
UCP3 stimulates glucose transport and GLUT4 translocation to the cell
surface in cardiac and skeletal muscle cells by activating a PI3K
dependent pathway.
Uncoupling proteins
(UCPs)1 are mitochondrial
inner membrane proteins proposed to be central to the regulation of
energy expenditure. Energy expenditure is composed of the resting
metabolic rate, physical activity, and thermogenesis and can be
increased by energy dissipation due to futile metabolic processes.
Uncoupling protein 1 (UCP1), the first uncoupling protein to be
identified, is selectively expressed in brown adipose tissue (BAT), a
major site of thermogenesis in rodents. UCP1 uncouples mitochondrial
respiration from ATP synthesis by dissipating the transmembrane proton
gradient, releasing energy as heat (1). The importance of UCP1 in
energy expenditure in adult humans is less clear, because little BAT is
present. Recently UCP2 and UCP3 were identified with 59 and 57% amino
acid identity to UCP1, respectively (2-4). UCP3, unlike
UCP1 and UCP2, exists as short and long form transcripts (5). The long
form of UCP3 was shown to be an uncoupling protein, because it
increases O2 consumption and decreases the mitochondrial
electrochemical potential when expressed in yeast or
C2C12 myoblasts (2, 4, 6, 7). Reconstitution of
UCPs into liposomes showed that UCP2 and UCP3, like UCP1, mediate
electrophoretic proton flux across lipid bilayers (8), providing
further evidence that these UCPs are functional uncoupling proteins.
UCP2 is expressed in many tissues, whereas UCP3
is expressed selectively in skeletal muscle in humans and primarily in
skeletal muscle and brown adipocytes in rodents with low levels in
white adipose tissue (3, 9). In humans, resting skeletal muscle metabolism is a significant determinant of whole body energy
expenditure and therefore is thought to play a role in body weight
regulation (10). Consistent with this, UCP3 gene expression
correlates negatively with body mass index and positively with sleeping
metabolic rate in Pima Indians (11). Furthermore, a polymorphism in the UCP3 gene is associated with reduced basal fat oxidation
rate and increased respiratory quotient in a specific African American population (12). Overexpressing either UCP3 or
UCP1 at very high levels in muscle of transgenic mice
results in increased energy expenditure, resistance to diet-induced
obesity, and increased glucose tolerance and insulin sensitivity (13,
14). However, recent studies with UCP3 knockout mice show
that, although endogenous UCP3 has uncoupling activity in skeletal
muscle mitochondria, the absence of UCP3 produces no apparent
abnormality in energy balance or glucose homeostasis (15, 16).
Interestingly, mitochondria lacking UCP3 produce more reactive oxygen
species (ROS) (15). Thus, the role of UCP3 in energy homeostasis and
fuel metabolism remains controversial.
Increased energy expenditure due to increased uncoupling activity is
expected to deplete energy stores and therefore raise fuel needs.
Because skeletal muscle is an important site for the regulation of
glucose disposal, increased uncoupling activity due to overexpression
of a UCP could result in increased glucose metabolism. In
the muscle-specific UCP1 and UCP3 overexpressing mice, fasting glucose is indeed lower and in epitrochlearis muscle from
UCP1 overexpressing mice the basal glucose uptake rate tends to be increased (13, 14). In humans, a positive correlation between
UCP3 mRNA and glucose utilization in lean NIDDM patients was recently demonstrated (17). However, these data are not yet
conclusive, because no such relationship was found in nondiabetic controls (17).
Glucose transport is rate-limiting for glucose metabolism. In L6
myotubes glucose uptake is stimulated 2- to 3-fold by insulin (18).
Treatment with the strong uncoupling agent dinitrophenol (DNP) for 30 min stimulates glucose uptake to the level of insulin in these cells,
and longer treatment stimulates above insulin-stimulated levels (19,
20). Long term treatment (18 h) with DNP also increases lactate levels
in L6 myotubes, because the cells cannot use oxidative phosphorylation
and therefore rely mainly on glycolysis to maintain ATP (18). These
findings indicate that L6 myotubes can adapt to situations of higher
energy demand such as uncoupling by increasing their glucose uptake and
metabolism. Furthermore, L6 cells express low endogenous levels of
UCP3, which can be metabolically regulated (21). Therefore, they are a
good model to study the effects of UCP3.
L6 muscle cells express the glucose transporters GLUT1, GLUT3, and
GLUT4 (22). Glucose uptake can be increased by inducing the expression
of one or more of these glucose transporters (GLUTs) or by eliciting
their redistribution from intracellular vesicles to the cell surface.
Insulin rapidly stimulates glucose transport in L6 cells as well as in
primary muscle and adipocytes by eliciting the translocation of GLUTs
to the cell surface via a signaling cascade requiring phosphoinositide
3-kinase (PI3K) (22). PI3K consists of an 85-kDa regulatory (p85) and a
110-kDa catalytic subunit (p110). The p85 subunit is normally a
cytosolic protein, but in response to insulin it translocates in
association with IRS proteins to specific intracellular membranes and
to the actin cytoskeleton (23). Chemical inhibitors of PI3K or a
dominant negative regulatory subunit of PI3K inhibit the
insulin-induced increase in glucose transport and glucose transporter
translocation (24-26). Overexpression of constitutively active PI3K
stimulates glucose transport, indicating that PI3K is sufficient for at
least part of the effect of insulin on glucose uptake (27). In
contrast, other agonists such as contraction, hypoxia, osmotic shock,
okadaic acid, and high dose DNP (0.5 mM) activate glucose
uptake by PI3K-independent mechanisms (19, 28-31).
In the present paper we studied the effects of UCP3 on glucose uptake
and metabolism and the underlying signaling pathways using two
complementary approaches. Human UCP3 (long form) was overexpressed in rat L6 myotubes by adenovirus-mediated gene transfer and in H9C2 cardiomyoblasts by stable
transfection with a tetracycline-repressible UCP3 construct.
Overexpression of UCP3 stimulates glucose transport and
GLUT4 recruitment to the cell surface to levels equivalent to the
effect of insulin, and the UCP3 effects are inhibited by wortmannin, LY
294002, or genistein. Furthermore, PI3K activity and p85 translocation
to membranes are increased in UCP3-overexpressing myotubes. Thus, UCP3
increases glucose transport and metabolism in cardiac and skeletal
muscle cells by a PI3K-dependent mechanism.
Cell Culture for L6 Myotubes, 3T3-L1 Adipocytes, and 293 Cells--
L6 muscle cells (gift from A. Klip, Toronta, Canada) were
grown and differentiated as described previously (32). Cells were used
for experiments 2-3 days after cell fusion, in the stage of myotubes.
3T3-L1 cells (ATCC, Rockville, MD, catalog no. CL-173) were grown and
differentiated as described previously (27). 293 cells were grown as
described for propagation of adenoviruses (27).
Generation of Recombinant Adenoviruses Encoding Human UCP3 or
HA-GLUT4--
The coding region of human UCP3 gene (5) was
removed as a Kpn-XbaI fragment and cloned into pACCMV.pLpA
(33), resulting in the plasmid pACCMV.pLpA-UCP3. The pCIS2 vector
containing the cDNA encoding the human GLUT4 with the
influenza virus hemagglutinin epitope (HA1) inserted in the first
exofacial loop of GLUT4 (34) (provided by S. Cushman,
Bethesda, MD) was digested to release the HA-GLUT4 cDNA
and cloned into pACCMV.pLpA, resulting in the plasmid
pACCMV.pLpA-HA-GLUT4. Recombinant adenoviruses were generated (27, 33).
Purification of recombinant virus by cesium chloride centrifugation
resulted in stocks of 1-2 × 109 pfu/ml for UCP3 and
3-4 × 1010 pfu/ml for HA-GLUT4 as determined by
limiting dilution. The recombinant adenovirus-encoding
Transduction of L6 Myotubes and 3T3-L1
Adipocytes--
Transduction of differentiated L6 myotubes and 3T3-L1
adipocytes was performed overnight with constant agitation on a rocking platform in
For GLUT4 translocation studies L6 myotubes were transduced with
UCP3 adenovirus as described above. After 14-h exposure
UCP3-encoding adenovirus was removed and L6 myotubes were
cotransduced with HA-GLUT4-encoding adenovirus at a
concentration of 1 × 107.
HA-GLUT4-encoding adenovirus was removed after 8-h exposure, and experiments were performed after an additional 40 h (total time of transduction for UCP3: 62 h and for
HA-GLUT4: 48 h).
Generation of H9C2 Cardiomyoblasts with
Repressible Expression of Human UCP3--
Polymerase chain reaction
was used to add AscI and NotI restriction enzyme
sites to the ends of human UCP3 (hUCP3) cDNA
(5) to facilitate cloning into a single-plasmid system (pKCTV) that was
constructed as described (35) for tetracycline repressible gene
expression in mammalian cells. DNA sequencing analysis confirmed the
correct hUCP3 coding sequence, and pKCTV/hUCP3 was
transfected into H9C2 cardiomyoblasts (ATCC)
plated at 60% confluency in 100-mm dishes using LipofectAMINE Plus
reagent (Life Technologies Inc.). Transfection was performed while
cells were growing in DMEM (high glucose), and serum was added 3 h
post-transfection to a final concentration of 10% fetal bovine serum
and 10% horse serum. Cells were split 2 days later into 150-mm dishes
and maintained in media containing hygromycin (400 ng/ml) for 2 weeks.
Colonies were isolated and screened for hUCP3 mRNA
expression with TaqMan quantitative reverse transcription-polymerase
chain reaction. Western blot analysis demonstrated protein expression
in hUCP3-expressing cells. hUCP3 mRNA and protein
expression were repressed by addition of doxycycline (5 ng/ml). To
induce UCP3 expression doxycycline was omitted. Wild-type
H9C2 cells were treated in the same way to control for potential nonspecific effects of doxycycline.
Survival of L6 Myotubes and H9C2 Cardiomyoblasts in Glucose-free
Media--
Transduced and nontransduced L6 myotubes were incubated in
the presence or absence of dinitrophenol (DNP, 0.5 mM) in
glucose-free Glucose Transport--
2-Deoxyglucose transport was determined
as previously described (27). DNP (0.5 mM) was added
during the 4-h serum-free incubation for the indicated times. Cells
were stimulated for 15 min with or without insulin (100 nM)
or DNP, then wortmannin (0.1 µM) or LY 294002 (100 µM) was added for 30 min in the continued presence or
absence of insulin. Adding insulin or DNP simultaneously with wortmannin or LY 294002 gave the same result. L6 myotubes and H9C2 cardiomyoblasts were incubated in the
presence or absence of genistein (300 µM) for 15 min and
left untreated or stimulated with insulin in the continued presence of
genistein for 30 min.
Lactate Production--
The medium was replaced at night, and
lactate released overnight into the medium was measured enzymatically
using a lactate kit (Sigma Diagnostics, St. Louis, MO).
Preparation of Cell Membranes or Lysates and Western
Blotting--
To generate a total membrane fraction and a cytosolic
fraction, L6 myotubes, H9C2 myoblasts, or
3T3-L1 adipocytes were homogenized by 20 strokes in a Potter
homogenizer and centrifuged at 180,000 × g for 75 min.
Proteins of the total membrane fraction were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (27). Membranes were blocked in Tris-buffered saline (TBS) with 0.05% Tween 20 and 5% low fat dry milk for 1 h
at room temperature. Membranes were incubated with primary antibodies against UCP3, p85 (
To generate total lysates of L6 myotubes, cells were treated as
described for PI3K assay (below). Proteins were separated by
SDS-polyacrylamide gel electrophoresis. Membranes were blocked in
Tris-buffered saline (TBS) with 0.05% Tween 20 and 5% low fat dry
milk or 4% bovine serum albumin (for antiphosphotyrosine) for 1 h
at room temperature. The nitrocellulose membranes were incubated with
polyclonal p85
Membranes were washed in TBS-0.05% Tween 20 (UCP3, GLUT3, p85,
phosphotyrosine, phospho-Akt) or TBS-0.5% Tween 20 (GLUT4); TBS-0.2%
Nonidet P-40; and subsequently TBS-0.1% Tween 20 (GLUT1) for 15 min
and incubated with horseradish peroxidase-coupled secondary antibodies
(27). Bands were visualized with enhanced chemiluminescence (Amersham
Pharmacia Biotech, Arlington Heights) and quantified by densitometry.
PI3K Assay--
PI3K activity in total lysates of L6 myotubes
was assayed using slight modifications of a protocol for muscle tissue
(37, 38). Following overnight incubation in Assay for Cell Surface Epitope-tagged GLUT4--
Translocation
of GLUT4 to the plasma membrane in L6 cells was assessed using a
modification of a protocol by Wang et al. (39). After 3-h
serum-free incubation wortmannin (0.1 µM) was added. Ten
minutes later, insulin (100 nM) was added for 20 min. Cells were quickly washed in ice-cold phosphate-buffered saline buffer and fixed in 3% paraformaldehyde for 3 min on ice. The fixative was
neutralized by incubation in 100 mM glycine in
phosphate-buffered saline for 10 min. Cells were blocked in 10% goat
serum for 10 min and incubated with horseradish peroxidase-conjugated
monoclonal HA-antibody from rat raised against the sequence YPYDVPDYA
of the HA protein (clone 3F10, Roche Molecular Biochemicals,
Indianapolis, IN) in a dilution of 1:100 for 60 min. Cells were washed
extensively and incubated with O-phenylenediamine dihydrochloride
reagent for 30 min protected from light. The reaction was
stopped by adding 3 M HCl. The supernatant was collected
and the absorbance was read at 492 nm in a microplate reader.
Statistical Analysis--
Statistical analysis was performed
using analysis of variance and Student's two-tailed t test
utilizing Statview software.
Expression of UCP3 in L6 Myotubes and 3T3-L1
Adipocytes--
Western blotting of total membranes showed a
moderately strong signal of 34 kDa in L6 myotubes transduced with
UCP3-encoding adenovirus and an even stronger signal in
3T3-L1 adipocytes transduced with UCP3 adenovirus. No signal
was detectable in control myotubes or adipocytes transduced with
UCP3 Increases Glucose Uptake in L6 Myotubes but Not in 3T3-L1
Adipocytes--
Insulin (100 nM) stimulated 2-deoxyglucose
uptake ~2-fold in nontransduced myotubes (Fig. 1B,
left panel) as reported (19). Expression of
Insulin stimulated glucose uptake about 13-fold in 3T3-L1 adipocytes
(Fig. 1B, right panel). In contrast to the
finding in L6 myotubes, UCP3 expression did not affect basal or
insulin-stimulated glucose uptake in 3T3-L1 adipocytes even though it
was expressed at a higher level than in L6 cells (Fig. 1A).
To further elucidate the lack of effect, we tested whether these
adipocytes could already be uncoupled to a high degree without UCP3
expression by incubating 3T3-L1 adipocytes with the uncoupling agent
DNP (0.5 mM) for 4 h. DNP treatment resulted in a
2-fold increase of glucose transport (DNP 19.8 ± 1.50 versus control 11.2 ± 0.99 pmol/min per well (p < 0.05); also reported in Ref. 18).
L6 Myotubes Overexpressing UCP3 Do Not Survive in Glucose-free
Media--
When cells are grown in glucose-free media they cannot use
glycolysis but rely on oxidative phosphorylation, which is blocked by
uncoupling. We tested the ability of L6 myotubes overexpressing UCP3 to survive in glucose-free media as an indication of
their state of uncoupling. Control cells that were not transduced (not shown) or were transduced with UCP3 Increases Lactate Production in L6 Myotubes--
Because Fig.
2 demonstrates the potential of UCP3-overexpressing cells to
generate energy from glycolysis, we hypothesized that UCP3
expression might result in higher conversion of pyruvate resulting from
glycolysis to lactate in normal media when glucose is present.
Expression of UCP3 increased lactate release into the media
1.5- to 2-fold above that in The Effects of UCP3 on Glucose Transport Are Mediated by
PI3K--
Wortmannin (0.1 µM, Fig.
4A) and LY 294002 (100 µM,
Fig. 4B) abolished insulin-stimulated glucose uptake in
nontransduced L6 myotubes and those transduced with
Fig. 5A shows that
UCP3 overexpression increased PI3K activity
immunoprecipitated with an antibody against phosphotyrosine 2.2-fold
compared with
To further elucidate the mechanism by which phosphotyrosine-associated
PI3K activity is increased in UCP3-overexpressing cells, we
determined both the level of the p85 regulatory subunit of PI3K and
tyrosine phosphorylation of cellular proteins in cell lysates of L6
myotubes. Expression of p85 was not increased in UCP3-expressing cells (data not shown). In addition,
UCP3 overexpression did not result in a detectable increase
in the tyrosine phosphorylation of any protein, in contrast to the
stimulatory effects of insulin on the phosphorylation of the insulin
receptor, IRS-1 and MAPK (data not shown). However, small changes in
tyrosine phosphorylation would be below the detection level of
antiphosphotyrosine immunoblotting. Thus, we used a more sensitive
assay, investigating the effect of the tyrosine kinase inhibitor,
genistein, on glucose uptake in UCP3-overexpressing
myotubes. Genistein (300 µM) completely abolished
insulin- and UCP3-induced glucose uptake (Fig. 5B), indicating that tyrosine phosphorylation is necessary for the effect of
UCP3 on glucose transport. UCP3 overexpression did not stimulate serine phosphorylation of Akt, a downstream target of PI3K
(Fig. 6). In contrast, Insulin (100 nM) markedly increased Akt phosphorylation in both
Expression of UCP3 Leads to Translocation of GLUT4 to the Plasma
Membrane in L6 Myotubes--
The total amount of endogenous GLUT1,
GLUT3, or GLUT4 was unchanged in UCP3-overexpressing L6
myotubes, as shown by immunoblotting of total membranes from these
cells (Fig. 7A). To determine
whether increased glucose transport was due to translocation of GLUT4 to the plasma membrane, we measured the amount of GLUT4 on the cell
surface after cotransducing L6 myotubes with adenoviruses encoding
Low Dose, Long Term Treatment with the Chemical Uncoupler DNP
Increases Glucose Uptake Also through a PI3K-mediated Pathway in
Contrast to High Dose, Short Term Treatment with DNP--
To determine
the specificity of the effects of UCP3 and whether other methods of
uncoupling also stimulate glucose transport through a
PI3K-dependent pathway, we studied the effect of short term
and long term treatment of cells with DNP. The effects of DNP (0.5 mM) to stimulate glucose transport are seen as early as 15 min (Fig. 8A). DNP exposure
for 30 min increased glucose uptake ~2-fold, similar to
insulin-stimulated levels. By 4 h, transport was increased
3.3-fold (Fig. 8B). Although there was a tendency for slight
inhibition of DNP-stimulated glucose transport by wortmannin after 30 min and 4 h of DNP treatment, the wortmannin effect was not
significant, in contrast to the complete inhibition of
insulin-stimulated glucose transport (Fig. 8C). However, a 10 to 50 times lower concentration of DNP (10-50 µM) for
the same duration as UCP3 overexpression (62 h) increased
glucose uptake up to 5-fold, and this effect was inhibited by
wortmannin (Fig. 8D). With the lower dose and longer
exposure to DNP, the level of lactate release was comparable to
UCP3 overexpressing cells (Fig. 8E) indicating a
similar degree of uncoupling. Thus, chronic, mild treatment with a
chemical uncoupler (DNP) also activates PI3K.
UCP3 Increases Glucose Uptake in H9C2 Cardiomyoblasts--
Fig.
9A shows that UCP3
expression was completely suppressed in H9C2
cardiac muscle cells stably transfected with human UCP3 (5)
under the control of a doxycycline-repressible promotor. Expression of
UCP3 was induced by omitting doxycycline from the medium.
After omission of doxycycline for 12 h, UCP3 expression was detected and expression progressively increased. After 7 days without doxycycline, UCP3 expression was 2.2-fold higher
than at 12 h of induction. The uncoupling effects UCP3 after
induction of UCP3 expression for 24 h and 7 days were
demonstrated by impaired cell survival when glucose was omitted from
the media (data not shown), similar to the effects in L6 cells in Fig.
2.
Fig. 9B shows that at 12 and 24 h of UCP3
induction, glucose transport was slightly increased by 1.3-fold
(p < 0.05) and 1.6-fold (p < 0.05),
respectively, compared with doxycycline-treated cells. 7 days after
induction when UCP3 expression was highest, glucose transport was increased about 2-fold over cells in which
UCP3 expression is not induced. The fact that insulin had
only a small effect on transport is not surprising, because these cells
were used as myoblasts. To control for any potential effects of
doxycycline, we measured glucose transport in wild-type
H9C2 cardiac myoblasts grown with and without
doxycycline for 12 h, 24 h, 48 h, and 7 days.
Doxycycline alone had no effect on glucose transport (data not shown).
Wortmannin (0.1 µM) completely abolished the increase in
glucose uptake in H9C2 cells in which UCP3 was induced for 7 days, at which time UCP3 expression and the stimulation of
transport was greatest (Fig. 9C). These data indicate that
activation of PI3K is necessary for the effect of UCP3 also in these
cardiomyoblasts. The tyrosine kinase inhibitor genistein (300 µM) also abolished the effect of UCP3
overexpression in H9C2 cardiomyoblasts (Fig. 9D), consistent with tyrosine phosphorylation being involved
in these effects.
UCP3 Expression Leads to Translocation of p85 and Increased PI3K
Activity in Total Membranes of H9C2
Cardiomyoblasts--
We prepared a total membrane fraction devoid of
cytosol but containing intracellular membranes (including mitochondrial
membranes) as well as plasma membranes. The amount of p85 appearing in
this fraction increased progressively with UCP3 expression
levels and was 5.5-fold higher after 7 days of UCP3
induction compared with noninduced cells (Fig.
10A). The total amount of
p85 in the cell homogenate remained unchanged (Fig. 10B).
Consistent with this, the PI3K activity in this fraction only 3 days
after induction of UCP3 expression increased 3.8-fold,
exceeding the effect of insulin (3.3-fold) in these cells (Fig.
10C). UCP3 stimulation of PI3K was completely inhibited by
wortmannin (0.1 µM) (Fig. 10D).
Mitochondrial uncoupling proteins alter membrane potential and
oxygen consumption and are proposed to play a major role in energy
expenditure (40). Although UCP3 knockout mice appear to have
normal glucose homeostasis (15, 16), high level overexpression of
either UCP1 or UCP3 in muscle of transgenic mice
results in increased energy expenditure, resistance to diet-induced
obesity, and increased glucose tolerance and insulin sensitivity (13, 14). This indicates that UCP3 may affect muscle cell glucose metabolism, although little is known about the mechanism for this or
the effects of increased UCP3 expression on intracellular
signaling pathways. This study demonstrates that UCP3
overexpression increases glucose transport in both cardiac and skeletal
muscle cells. This effect results from increased recruitment of GLUT4
to the cell surface and is dependent on activation of PI3K.
Most existing data on the role of uncoupling proteins in glucose
utilization are correlative. Cold exposure stimulates glucose uptake in
brown adipose tissue (BAT) of normal rats (41). Because cold exposure
activates UCP1 expression in BAT via the sympathetic nervous
system, the effect on glucose transport may involve increased UCP1
activity, but this needs to be tested. Recently, Krook et al. (17) demonstrated a positive correlation between
UCP3 mRNA and glucose utilization in lean NIDDM
patients. Although no such relationship was found in nondiabetic
controls, the data suggest a relationship between UCP3
expression and glucose homeostasis at least under some conditions.
Although glucose oxidation is normal in UCP3 knockout mice
(15, 16), this could be due to compensatory alterations during
development. Further studies are needed to determine whether glucose
utilization is normal under all metabolic conditions.
Cellular stressors such as contraction/exercise, hypoxia, osmotic
shock, and heat shock (19, 28-31), which increase energy demands
stimulate glucose transport. Most of these manipulations result in
decreased ATP levels, and the concomitant increase in glucose transport
has been interpreted to be a response to generate ATP via glycolysis.
With strong uncoupling agents such as DNP, the increase in glucose
transport persists even though ATP levels are rapidly restored (18). In
contrast to our current findings, stimulation of glucose transport in
all of these stress situations, including short term, high dose DNP
treatment, does not depend on signal transduction via the PI3K pathway
(19, 28-31). However, high dose DNP has much stronger uncoupling
effects than UCP3 when overexpressed at the levels in our study (20).
Consistent with this is the shorter cell survival in glucose-free media
of cells treated with high dose DNP compared with cells overexpressing UCP3 (Fig. 2). DNP treatment at a 10 to 50 times lower concentration for as long as the UCP3 overexpression lasted (62 h) also
increased glucose uptake, but this effect was inhibited by wortmannin.
The levels of lactate release from UCP3-overexpressing cells
and from low dose, long term DNP-treated cells were comparable,
suggesting the same degree of uncoupling. Thus, not only UCP3, but
other methods of mild, long term uncoupling activate a signaling
cascade through PI3K.
We found a 2.2-fold increase in PI3K activity in L6 cell lysates and a
3.8-fold increase in membranes of H9C2 cells.
Most likely this difference reflects the high association of activated PI3K with membranes. The mechanism by which increased uncoupling activity could increase PI3K activity is of interest. UCP2 and UCP3
play a role in the regulation of mitochondrial ROS generation (15, 42,
43). The effect of UCP3 could be mediated by altering levels of
reactive oxygen species (ROS). A growing body of evidence suggests a
potential role for oxygen-derived radicals such as hydrogen peroxide
and superoxide anions as intracellular signaling molecules (for review
see Ref. 44). Mitochondrial metabolism of pyruvate activates the c-Jun
N-terminal kinase (JNK), which is triggered by increased release of
mitochondrial H2O2, leading to inhibition of
glycogen synthase 3 Our findings raise the possibility that PI3K may be activated in
association with mitochondrial function in muscle. This may be indirect
via other cellular signals, which are activated by mitochondrial
uncoupling such as ROSs, or via direct physical interaction of PI3K at
the site of mitochondria. No data are published about the presence or
activation of PI3K activity in mitochondria. However, PI3K is
stimulated in nuclei of leukemia cells during granulocyte
differentiation (50). Furthermore, a phosphatidylinositol 3,4,5-triphosphate binding protein localizes in the nucleus in brain
(51) and hydrogen peroxide treatment leads to PI3K translocation to the
nucleus where its activity is enhanced. Further studies will determine
whether this occurs in mitochondria.
In the current study, we found that the inhibitor of tyrosine
phosphorylation, genistein, inhibited the stimulatory effect of UCP3 on
glucose transport (Figs. 5B and 9D). However, we
did not detect any obvious increase in tyrosine phosphorylation by antiphosphotyrosine immunoblotting. It is likely that the level of
phosphorylation change required for the degree of stimulation of
phosphotyrosine-associated PI3K activity in
UCP3-overexpressing L6 cells(Fig. 5A) would be
below the detection limit of Western blotting. The lack of effect on
Akt is not surprising, given that we and others found a discordance
between PI3K activity and Akt stimulation in other models (as reviewed
in Ref. 37).
The effects of UCP3 on glucose transport appear to be tissue-specific,
because they are seen in cardiac and skeletal muscle cells but not in
adipocytes. This may result from the fact that adipocytes are already
more uncoupled than muscle cells. Strong uncoupling with DNP treatment
stimulates glucose transport to only ~15% of the maximal effect of
insulin in adipocytes, whereas it reaches the maximal effect of insulin
in muscle cells (see "Results" and Ref. 18). Thus, adipocytes have
the potential for greater glucose transport stimulation but uncoupling
has minimal effect. Because in humans UCP3 is expressed
selectively in muscle, we speculate that regulation of human UCP3
activity requires muscle-specific factors.
In summary, these data demonstrate that UCP3 overexpression
increases glucose transport and metabolism in skeletal and cardiac muscle cells. Furthermore, these effects of UCP3 appear to involve GLUT4 translocation and are mediated by activation of PI3K, because UCP3 overexpression increases PI3K activity and the effect
of UCP3 to stimulate glucose transport and GLUT4 cells surface
recruitment are blocked by wortmannin and LY 294002. These findings
indicate that mitochondrial uncoupling activity could play an important role in cellular glucose utilization and that uncoupling proteins can
activate intracellular signal transduction pathways with a multiplicity
of effects on cellular physiology and metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase was provided by C. Newgard and amplified
(27).
-MEM with 2% fetal bovine serum or DMEM with 10% fetal
calf serum respectively. UCP3 or
-galactosidase-encoding recombinant
adenoviruses were used at a concentration of 1 × 108
pfu/ml (as determined by limiting dilution) for L6 myotubes and 1 × 109 pfu/ml for 3T3-L1 adipocytes. Virus was removed
after 14-h exposure, and experiments were performed following an
additional 48-h incubation in fresh media.
-MEM with 2% fetal bovine serum or in standard
-MEM
with 25 mM glucose and 1 mM pyruvate.
Similarly, H9C2 myoblasts treated with or without doxycycline for
24 h and 7 days were grown in glucose-free DMEM or in standard
DMEM. The media with or without DNP was changed daily. Survival or cell
death was documented by photography using an inverted microscope (Nikon
AFX-DX) equipped with a 35-mm camera system.
-subunit), and GLUT1, GLUT3, or GLUT4 glucose transporters overnight. Anti-human UCP3 antiserum (1:1000) was raised
against a peptide representing residues 146-167 of human UCP3 and was
affinity-purified by coupling the same peptide to Pierce SulfoLink gel.
Polyclonal GLUT1 antiserum (1:100) was provided by B. Thorens
(Lausanne, Switzerland); polyclonal anti-mouse GLUT3 (36) (1:600),
provided by I. A. Simpson (Bethesda, MD); polyclonal anti-GLUT4
(1:400), provided by H. Haspel (Detroit, MI); and monoclonal p85
antibody (
-subunit) (1:1000) was purchased from Upstate
Biotechnology Inc. (Lake Placid, NY).
antiserum (Upstate Biotechnology Inc.) at 1 µg/ml,
monoclonal anti-phosphotyrosine (Santa Cruz Biotechnology, Santa Cruz,
CA) at a dilution of 1:200, and polyclonal anti-serine-phosphorylated
Akt (serine 473, New England BioLabs, Beverly, MA) at
1:1000.
-MEM containing 0.1% calf serum, L6 myotubes were or were not stimulated with 100 nM insulin for 10 min, lysed for 30 min at 4 °C in lysis
buffer (37), and centrifuged for 10 min at 14,000 rpm to remove unlysed
debris. Total lysates (200 µg of protein) were subjected to
immunoprecipitation with monoclonal antiphosphotyrosine antibody (4G10)
(1:100 dilution; gift from C. R. Kahn, Joslin Diabetes Center),
and the assay was carried out as described (37). Total membranes of
H9C2 cardiomyoblasts were obtained as described
above and subjected to immunoprecipitation with monoclonal
antiphosphotyrosine antibody (py99) (1:50 dilution from Santa Cruz
Biotechnology). The assay was carried out as described (37).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (Fig. 1A).
The expression of UCP3 per milligram of membrane protein in 3T3-L1
adipocytes is ~2-fold higher than in L6 myotubes when considering the
different protein amounts loaded on the gel for adipocytes (25 µg/lane) compared with myotubes (12 µg/lane). We routinely use 10×
higher concentration of adenovirus for 3T3-L1 adipocytes (see Fig. 1 legend) compared with L6 myotubes to achieve
90% of cells transduced (27).
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Fig. 1.
A, expression of human UCP3
in L6 myotubes and 3T3-L1 adipocytes. Cells were transduced overnight
with recombinant adenoviruses encoding -galactosidase or human
UCP3 (~1 × 108 pfu/ml for myotubes and
~1 × 109 pfu/ml for adipocytes). Following an
additional 48-h incubation, total membranes were prepared and
immunoblotted for UCP3 as described under "Experimental Procedures"
(12 µg/lane for myotubes and 25 µg/lane for adipocytes). The blots
represent four independent experiments. B, UCP3 increases
2-deoxyglucose uptake in L6 myotubes but not in 3T3-L1 adipocytes.
Cells were not transduced (no virus) or were transduced overnight with
adenoviruses encoding
-galactosidase or
hUCP3. After an additional 48-h incubation, which included
4 h of serum-free incubation, 2-deoxyglucose uptake was determined
as described under "Experimental Procedures." Data for L6 myotubes
are expressed as relative stimulation over basal levels in no virus
cells and are means ± S.E. of 7-9 independent experiments, each
performed in triplicate. Data for 3T3-L1 adipocytes are representative
of three total experiments and are means ± S.E. of triplicates.
*, different from no virus and
-galactosidase basal at
p < 0.05.
-galactosidase did not significantly change basal or
insulin-stimulated glucose uptake. In contrast, expression of
UCP3 increased glucose uptake ~2-fold, thereby reaching
maximally insulin-stimulated levels in control cells. Insulin did not
significantly increase glucose uptake further in UCP3 expressing L6
myotubes although there was a tendency for a further increase.
-galactosidase (Fig.
2A) and grown in glucose-free
media survived for at least 6 days without alteration in morphology
before they were discarded. In contrast, UCP3-overexpressing myotubes lost their tubular structure and died after 2-3 days in
glucose-free media (Fig. 2B), whereas the same
UCP3-overexpressing cells maintained in the presence of 25 mM glucose survived morphologically unchanged with
prominent tubular structure (Fig. 2C). The death of
UCP3-overexpressing cells in glucose-free media (Fig.
2B) is consistent with UCP3 having uncoupling activity (6,
7) and thereby decreasing oxidative phosphorylation in L6 myotubes. For comparison we treated myotubes in parallel wells overnight with DNP.
All DNP-treated cells, including those nontransduced or transduced with
-galactosidase or UCP3, died (not shown), consistent with the fact that the cells are dependent on glycolysis. Furthermore, the
more rapid death with DNP incubation (i.e. overnight)
suggests that the uncoupling activity of UCP3 is lower than the
activity of DNP used at 0.5 mM.
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Fig. 2.
Effects of UCP3 expression
on cell survival in glucose-free media. L6 myotubes were not
transduced (no virus) or were transduced with adenoviruses (see Fig.
1). 48 h later, cells in A and B were
incubated in glucose-free media. Photographs were taken on day 3 of
incubation in glucose-free media. A, cells transduced with
-galactosidase; B, cells transduced
with UCP3; C, cells transduced with
UCP3 grown in media containing 25 mM
glucose.
-galactosidase-transduced and
nontransduced cells (Fig. 3).
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Fig. 3.
UCP3 increases release of lactate into media
by L6 myotubes. Cells were not transduced (no virus) or were
transduced with adenoviruses (see Fig. 1). After an additional 48 h, including a media change 16 h before measurement, lactate
levels in the media was determined. Data represent the means ± S.E. of six independent experiments. *, different from no virus and
-galactosidase basal at p < 0.05.
-galactosidase
as expected. Furthermore, wortmannin and LY 294002 completely abolished
the increase in glucose uptake in myotubes overexpressing
UCP3, indicating that activation of PI3K is necessary for
the effect of UCP3.
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Fig. 4.
A and B, wortmannin
(A) and LY 294002 (B) abolish the effect of UCP3
to increase glucose transport in L6 myotubes. Cells were not transduced
(no virus) or transduced with adenoviruses (see Fig. 1). 48 h
later, cells were incubated for a total of 45 min in the presence or
absence of insulin (100 nM). Wortmannin (0.1 µM) (A) and LY 294002 (100 µM)
(B) were added for the last 30 min and 2-deoxyglucose uptake
was determined. *, different from no virus and -galactosidase basal
at p < 0.05.
-galactosidase-transduced cells (p < 0.05). We also immunoprecipitated proteins in the cell lysates with
preimmune serum as background control for PI3K activity. The background activity of PI3K was less than 10% of the activity immunoprecipitated with phosphotyrosine antibody, and thus negligible. Insulin increased PI3K activity ~7-fold in control cells transduced with
-galactosidase (data not shown). Both the effect of insulin and
UCP3 overexpression on PI3K activity were completely
inhibited by wortmannin (0.1 µM) (data not shown).
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Fig. 5.
A, PI3K activity is increased in L6
myotubes overexpressing UCP3. L6 myotubes were not
transduced (no virus) or transduced with adenoviruses (see Fig. 1).
After an additional 48-h incubation, including overnight serum
starvation, cells were incubated for 10 min in the presence or absence
of insulin (100 nM) and lysed. PI3K activity was measured
in antiphosphotyrosine immunoprecipitates. This blot is representative
of four independent experiments. The bar graph shows
means ± S.E. of densitometric quantitation of four experiments.
*, different from -galactosidase cells at p < 0.05. B, genistein inhibits UCP3-stimulated glucose uptake in L6
myotubes. Cells were transduced as described above. After 4 h of
serum-free incubation, genistein (300 µM) was added for
15 min followed by a 30-min incubation with insulin (100 nM) in the continued presence of genistein, and
2-deoxyglucose uptake was determined. *, different from basal at
p < 0.05. In all panels inhibition of insulin- or
UCP3-stimulated glucose uptake by wortmannin, LY 294002, or genistein
was significant at p < 0.05.
-galactosidase- and UCP3-transduced cells.
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Fig. 6.
Serine phosphorylation of Akt is not
increased in L6 myotubes overexpressing UCP3. L6 myotubes were
treated as in Fig. 5A. After overnight serum starvation,
cells were incubated for 10 min in the presence or absence of insulin
(100 nM), lysed, and analyzed by Western blotting with an
antibody against serine-phosphorylated Akt. The blot
represents results from two independent experiments.
-galactosidase or UCP3 and with
HA-GLUT4-encoding adenovirus (Fig. 7B). Insulin
(100 nM) increased the amount of HA-GLUT4 on the cell
surface 2- to 2.3-fold over basal levels in nontransduced cells and
-galactosidase-transduced cells cotransduced with
HA-GLUT4. This effect was completely inhibited by wortmannin
(0.1 µM). Expression of UCP3 caused a 2.3-fold
increase of HA-GLUT4 on the cell surface, which was also completely
blocked by wortmannin (0.1 µM).
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Fig. 7.
A and B, expression of
UCP3 leads to translocation of GLUT4 to the plasma membrane
but not to increased expression of GLUT1, 3, or 4 in L6 myotubes.
A, L6 myotubes were not transduced (no virus) or were
transduced with adenoviruses (see Fig. 1). 48 h later, total
membranes were prepared and immunoblotted for GLUT1, GLUT3, and GLUT4.
Values represent densitometry units/µg protein and are means ± S.E. of three independent experiments. B, L6 myotubes were
transduced with UCP3 adenovirus as described for Fig. 1.
After removing the UCP3-encoding adenovirus, L6 myotubes
were cotransfected with HA-GLUT4-encoding adenovirus at a
concentration of 1 × 107. Virus was removed after 8-h
exposure, and experiments were performed after an additional 40 h
(total time of transduction for UCP3 was 62 h and for
HA-GLUT4 was 48 h). Cell surface-bound HA-GLUT4 was
detected using a horseradish peroxidase-conjugated HA antibody on
nonpermeabilized cells, which were fixed in paraformaldehyde. Values
represent arbitrary units/well of a 24-well plate and are means ± S.E. of six to seven determinations. *, different from basal at
p < 0.01.
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Fig. 8.
A, B, and C,
wortmannin abolishes the effect of low dose, long term DNP treatment
but not high dose, short term DNP treatment to increase glucose
transport in L6 myotubes. A, cells that were not transduced
were incubated for 4 h in serum-free media in the presence or
absence of DNP (500 µM) for the indicated times.
2-Deoxyglucose uptake was determined. B, cells that were not
transduced were stimulated with DNP (500 µM) for 30 min
or 4 h or insulin (100 nM) for 30 min in the presence
or absence of wortmannin (0.1 µM). C, cells
that were not transduced were incubated for 64 h in the presence
of DNP at the indicated concentrations, media was changed daily.
Wortmannin (0.1 µM) was added 30 min prior to measuring
2-deoxyglucose uptake. D, low dose, long term DNP treatment
increases lactate release from L6 myotubes to the same extent as
overexpression of UCP3. Media was changed to serum free -MEM after
60 h of DNP treatment, and lactate levels in the media were
determined 4 h later. Data represent means ± S.E. of three
to five independent experiments, each performed in triplicate. *,
different from basal at p < 0.05; #, different from
insulin at p < 0.05.
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Fig. 9.
A, induction of UCP3
expression in H9C2 cardiomyoblasts.
H9C2 myoblasts were stably transfected with
UCP3 under the control of a doxycycline repressible
promotor. After the indicated incubation time with or without
doxycycline (5 ng/ml) total membrane fractions were prepared and
analyzed by Western blotting with an antibody against human UCP3. This
Western blot represents results from three independent experiments.
B, UCP3 expression increases 2-deoxyglucose
uptake in H9C2 myoblasts. UCP3
expression was induced as in A. After 4-h serum starvation
in the presence or absence of insulin (100 nM) for 30 min,
2-deoxyglucose uptake was determined. Data are means ± S.E. of
triplicates and are representative of three independent experiments. *,
values from basal and insulin-stimulated cells at 12 h, 24 h,
and 7 days of UCP3 induction are different from basal
noninduced at p 0.05; #, insulin-stimulated
value in 7 days induced cells is different from insulin-stimulated
noninduced cells at p < 0.02. C, wortmannin
inhibits UCP3-stimulated glucose uptake in H9C2
myoblasts. Cells were treated as described in B and were
incubated in the presence or absence of insulin for 30 min with or
without wortmannin (0.1 µM). These data represent results
from three independent experiments. *, different from basal at
p < 0.05; #, insulin-stimulated value in 7 days
induced cells versus noninduced cells is different at
p < 0.05. D, genistein inhibits
UCP3-stimulated glucose uptake in H9C2 myoblasts. UCP3
expression was induced as in A. After 4 h of serum-free
incubation genistein (300 µM) was added for 15 min
followed by a 30-min incubation with insulin (100 nM) in
the continued presence of genistein, and 2-deoxyglucose uptake was
determined. *, different from basal at p < 0.05. In
all panels inhibition of insulin- or UCP3-stimulated glucose uptake by
wortmannin or genistein was significant at p < 0.05.
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Fig. 10.
A and B, overexpression of
UCP3 leads to translocation of p85 to total membranes of
H9C2 myoblasts UCP3 expression was
induced as in A. Homogenate and total membranes were
obtained as described under "Experimental Procedures" and analyzed
by Western blotting using antibodies against p85 ( -subunit) or UCP3.
30 µg of protein per lane were loaded for homogenate and 20 µg per
lane for total membrane. Blots are representative of two to
three independent experiments. The bars represent the
average of densitometric quantitation of two to four experiments. *,
different from basal at p < 0.05. C. PI3K activity is
increased in total membranes of H9C2 myoblasts
overexpressing UCP3. UCP3 expression was induced
for 72 h as described under "Experimental Procedures." After
4 h of serum-free incubation, cells were incubated for 10 min in
the presence or absence of insulin (100 nM) with or without
wortmannin (0.1 µM), and total membranes were obtained by
centrifugation as described under "Experimental Procedures." PI3K
activity was measured in antiphosphotyrosine immunoprecipitates. The
bar graph shows means ± S.E. of densitometric
quantitation of three experiments. *, different from basal at
p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and activation of glycogen synthase (45).
Interestingly, stimulation of vascular smooth muscle cells with
platelet-derived growth factor results in increased ROS. When ROSs are
rapidly removed with scavenger compounds, the tyrosine phosphorylation
effects of platelet-derived growth factor are abolished (46). In
addition, the reactive oxygen species hydrogen peroxide alters both
basal and insulin-stimulated glucose transport in myotubes and
adipocytes (47-49) by a PI3K-dependent mechanism (49).
These studies support the possibility that ROSs alter signaling
pathways involving tyrosine phosphorylation and PI3K.
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ACKNOWLEDGEMENTS |
---|
We thank A. Klip and P. Bilan for L6 myoblasts and for helpful advice, C. Newgard for adenovirus plasmids, S. Cushman for the HA-GLUT4 plasmid, I. Simpson and D. Yver for GLUT3 antiserum, C. R. Kahn for phosphotyrosine antiserum, H. Haspel for GLUT4 antiserum, B. Thorens for GLUT1 antiserum, and C.-Yu Zhang and C. Sundberg for stimulating discussions and experimental advice.
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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants DK 43051 (to B. B. K.) and DK 49569 (to B. B. L.) and a grant from Eli Lilly.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by research fellowships from Eli Lilly/European Association for the Study of Diabetes and the American Diabetes Association.
** To whom correspondence should be addressed: Diabetes Unit, Research North 325E, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-5422; Fax: 617-667-2927; E-mail: bkahn@caregroup.harvard.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M011708200
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ABBREVIATIONS |
---|
The abbreviations used are:
UCP, uncoupling
protein;
PI3K, phosphoinositide 3-kinase;
DNP, dinitrophenol;
IRS-1, insulin receptor substrate-1;
IR, insulin receptor;
BAT, brown adipose
tissue;
ROS, reactive oxygen species;
GLUT, glucose transporter;
pfu, plaque-forming unit(s);
DMEM, Dulbecco's modified Eagle's medium;
MAPK, mitogen-activated protein kinase;
JNK, c-Jun N-terminal kinase;
-MEM,
minimal essential medium.
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REFERENCES |
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