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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 24, 2001 as doi:10.1096/fj.00-0828fje. |
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Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, 08028 Barcelona, Spain; and
* Nestlé Research Center, Vers chez Lausanne les 26 Blanc 1000, Switzerland
3Correspondence: Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franquès, 1, 08028 Barcelona, Spain. E-mail: anamaria{at}sun.bq.ub.es
SPECIFIC AIM
Uncoupling protein 3 (UCP3) is believed to act as a proton/anion carrier, but little is known of its metabolic function. In this study, the effect of UCP3 overexpression on nucleotide levels and nutrient oxidation in primary cultures of human muscle cells was examined.
PRINCIPAL FINDINGS
1. Overexpression of UCP3 lowers the mitochondrial membrane
potential
Treatment of muscle cells with a recombinant adenovirus containing
the full cDNA for the long form (L-UCP3)UCP3 protein (AdCMV-UCP3) led
to a time-dependent increase in UCP3 protein in the mitochondrial
fraction, whereas in control cells UCP3 expression could be detected
only by semiquantitative RT-PCR. Ectopic expression of UCP3 did not
cause changes in the expression of the UCP2 gene.
Mitochondrial membrane potential (
m) was estimated as the uptake
of the JC-1 dye and was expressed as a percentage of the value in
control cells incubated with glucose. In control cells, m
differed depending on the substrate provided to cells; it was higher in
cells incubated with oleate (141±12) than with glucose, in agreement
with the previously reported effect of fatty acids. When
glucose-incubated cells were treated with the protonophoric uncoupler
CCCP, m was dramatically reduced (90%), as expected.
Overexpression of UCP3 decreased m in cells incubated with
glucose (68±8) or oleate (77±7).
2. UCP3 lowers ADP and raises ATP/ADP ratio
The total content of nucleotides was not modified by the
overexpression of UCP3. Whereas no significant changes in ATP
and AMP contents were observed in UCP3-overexpressing cells vs.
controls, a marked decrease in the ADP content was detected. ADP levels
50% and 40% lower than in controls in glucose or oleate-incubated
cells. In UCP3-overexpressing cells, the ATP/ADP ratio was 150% that
in controls when provided with either glucose or oleate. The effect of
UCP3 on nucleotide content was compared with that of the chemical
uncoupler CCCP. In glucose-incubated cells, treatment with 5 µM
CCCP for 1 h resulted in a marked reduction in the ATP content
(CCCP-treated cells: 35±1 nmol ATP/mg protein; control cells 52±5
nmol ATP/mg protein) whereas the ATP/ADP ratio was also decreased to
3.3 ± 0.3 vs. 5.4 ± 0.5.
3. Oxidation of fatty acids is stimulated in UCP3-overexpressing
cells regardless of glucose presence
To analyze the effect of UCP3 overexpression on fatty acid
oxidation, cells were preincubated in the absence of glucose and
further incubated with 50 µM [14C]oleate
without glucose or 25 mM glucose. The amount of
[14C]-CO2 released was
quantified (Fig. 1A
). When compared with control cells, a higher yield of
[14C]-CO2 was detected in
UCP3-overexpressing cells preincubated without glucose (increase of
33%) and with glucose (increase of 26%). Glucose exerted a
marked inhibitory effect on fatty acid oxidation that was equivalent in
both cell types and led to a decrease of 45% in
[14C]-CO2 production.
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To ascertain whether the stimulation of fatty acid oxidation in
UCP3-overexpressing cells was dependent on CPTI activity, fatty
acid oxidation was measured in the presence of a CPTI activator
(L-carnitine) and a CPTI irreversible inhibitor (etomoxir).
Supplementation with L-carnitine markedly increased the oxidation of
[14C]-oleate in both control and
UCP-3-overexpressing cells by 2.5-fold, whereas no modification in
the extent of UCP3 stimulation was found. Treatment of cells with
etomoxir decreased [14C]-oleate oxidation in
control and UCP3-overexpressing cells by 70%. Notably, the
stimulation of fatty acid oxidation by UCP3 was suppressed in
etomoxir-treated cells.
4. Oxidation of glucose is stimulated in UCP3-overexpressing cells
whereas fatty acid-mediated inhibition is enhanced
Glucose oxidation was assessed by determining the
[14C]-CO2 production from
[14C]-glucose in cells incubated in the
presence or absence of 0.5 mM oleate (Fig. 1B
).
Overexpression of UCP3 led to an increase of 27% in the yield of
[14C]-CO2 compared with
control cells incubated with glucose alone. Addition of oleate did not
significantly modify the oxidation rate of glucose in control cells,
indicating that in this condition the glucose-fatty acid cycle was not
operative. In contrast, the stimulation of glucose oxidation caused by
UCP3 was inhibited in the presence of oleate, indicating that
UCP3-overexpressing cells gained sensitivity to the fatty acid
inhibitory effect on glucose oxidation.
5. Effects of UCP3 on glucose uptake and the glycolytic flux
Experiments were undertaken to determine whether glucose uptake
and disposal through glycolysis was enhanced by UCP3 overexpression.
2-Deoxyglucose uptake in AdCMV-UCP3-treated cells was higher (28.1±1.2
pmol/min per well) than in cells treated with the control virus
(19.3±1.2 pmol/min per well). The flux through the glycolytic pathway
was estimated as the flux through the 6-phosphofructo-1-kinase as
assessed by the release of
3H2O from
[5-3H]-glucose. The yield of
3H2O from
[5-3H]-glucose was slightly higher in
AdCMV-UCP3-transduced cells (24.5±1.6 nmol glucose/4 h mg protein)
than in control cells (19.5±1.6 nmol glucose/4 h mg protein). When
cells were incubated with 5 µM CCCP, the release of
3H2O (32.4±1.7 nmol
glucose/4 h mg protein) was much higher than in controls. Lactate and
pyruvate release was measured in cells incubated with 25 mM glucose
DMEM for 48 h. Lactate concentration in the media from
AdCMV-UCP3-transduced cells (30±0.8 mM) was not different from that of
control cells (28±0.5 mM). In contrast, the amount of released
pyruvate was reduced in UCP3-overexpressing cells (0.041±0.005 mM) as
compared with controls (0.086±0.002 mM). Therefore, the
lactate/pyruvate ratio, which is an indicator of the cytosolic
NADH/NAD+ ratio, was twice as high in the
AdCMV-UCP3-treated cells (631±70) as in controls (323±8). In control
cells incubated for 8 h with 5 µM CCCP, lactate levels (40±0.8
mM) were already 40% higher than in untreated cells, whereas pyruvate
concentration was markedly reduced (0.036±0.003 mM). As a result, the
lactate/pyruvate ratio rose to 965 ± 12 after treatment with
CCCP.
CONCLUSIONS AND SIGNIFICANCE
When UCP3 was overexpressed in cultured human skeletal muscle cells, it led to a decrease in the mitochondrial membrane potential, although the effect was less than that of the protonophoric uncoupler CCCP. Remarkably, UCP3 differed from the chemical uncoupler in that the proton leak was accompanied by a decrease not in the ATP content of cells, but in ADP. The ATP/ADP ratio was increased in UCP3-overexpressing cells in contrast to the decrease induced by CCCP. On the other hand, UCP3 exhibited a characteristic effect of chemical uncouplers: to increase the lactate/pyruvate ratio, an indicator of the cytosolic NADH/NAD+, apparently by impairing the malate-aspartate shuttle.
UCP3 overexpression stimulated both fatty acid and glucose oxidation, but differentially affected their competitive oxidation. Oxidation of oleate was increased in UCP3-overexpressing cells and this effect appeared to be dependent on the entry of Acyl-CoA into the mitochondria through CPTI, since it was abolished by addition of etomoxir, an irreversible CPTI inhibitor. However, UCP3-dependent stimulation was exerted regardless of the presence or absence of glucose. Glucose markedly reduced oleate oxidation in accordance with the operation of the glucose-fatty acid cycle, where inhibition of CPTI by a glucose-dependent increase in malonyl CoA is proposed. Therefore, from our data we can conclude that partial inhibition of CPTI by glucose still allows the effect of UCP3 to be manifested.
UCP3 also increased glucose oxidation, although this effect was inhibited by provision of fatty acid. This was an important finding since in control cells, no inhibition of glucose oxidation by oleate could be detected, indicating no operation of the fatty acid-glucose cycle as observed in muscle in vivo. In contrast, in UCP3-overexpressing cells the fatty acid inhibitory effect was revealed, suggesting that UCP3 may collaborate to inhibit the flux through pyruvate dehydrogenase. Nevertheless, through this putative mechanism, UCP3 would contribute to favor fatty acid vs. glucose oxidation, thus regulating nutrient partioning.
The metabolic effects attained by UCP3 overexpression were modest
despite the large increase in the protein, but this agrees with the
observation that a very high overexpression of UCP3 in transgenic mice
was required to develop a metabolic phenotype. Here we show that UCP3
metabolic effects cannot be attributed to an increase in the ATP/AMP
ratio, which is known to be a crucial regulator of fatty acid and
glucose oxidation through activation of AMP-kinase or allosteric
effects. Consistently, we show weak effects of UCP3 on the glycolytic
pathway that differ markedly from the powerful stimulation caused by
the chemical uncoupler CCCP, which is associated with the decrease in
the ATP/ADP ratio. Rather, UCP3 effects on oxidation appear to rely on
the changes associated with a decrease in m, such as increased
respiratory chain activity and decreased NADH translocation to the
mitochondria (Fig. 2
). We propose that this differential effect, which questions the
consideration of UCP3 as an uncoupler of oxidative phosphorylation,
provides a biological significance to UCP3 that will not cause highly
stimulated glycolysis and glucose consumption when up-regulated in
metabolic stress situations. Furthermore, we show that fatty
acid-mediated inhibition of glucose oxidation is enhanced by UCP3, so
that mitochondrial oxidation of acyl-CoA is primed with respect to the
oxidation of glucose. This observation may be related to the notion
that UCP3, besides promoting nutrient oxidation, particularly favors
fatty acid usage.
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FOOTNOTES
1 To read the full text of this article, go
to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0828fje ; to
cite this article, use FASEB J. (July 24, 2001)
10.1096/fj.00-0828fje 
2 Present address: Laboratoire de Physiologie et
Regulations Energétiques, Cellulaires et Moleculaires,
Université Claude Bernard-LyonI, 43, Bld. 11 Novembre 1918,
69622, Villeurbanne, France.
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) or AdCMV-UCP3 virus (
) were
preincubated for 18 h in the absence of glucose and oleate. They
were further incubated for 4 h with A) 50 µM
[1-14C]-oleate in the presence of 2 mM L-carnitine, 40
µM etomoxir, or 25 mM glucose or with no other additions (control) or
B) 10 mM [U-14C]-glucose in the presence
or absence of 0.5 mM oleate. At the end of this period, the yield of
[14CO2] was quantified. Results are expressed
as pmol of oxidized [1-14C]-oleate or
[U-14C]-glucose/well and are mean ±
SE from 3 experiments performed in quadruplicate
dishes The significance of the differences between controls and
UCP3-overeexpressers are *P < 0.05; **P
< 0.01; ***P < 0.001.