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,1
* Université Paris Descartes, Faculté de Médecine site Necker, Paris, France;
Biomedical Research Building II/III, Philadelphia, Pennsylvania, USA
1Correspondence: Université Paris Descartes, CNRS-UPR9078, Faculté de Médecine site Necker, 156 rue de vaugirard, 75730 Paris Cedex 15, France. E-mail: pecqueuc{at}necker.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: carrier • mitochondria • metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. UCP1 has been clearly established as the molecular mediator of nonshivering thermogenesis (2)
. The structural resemblance of UCP2 to UCP1, its ability to uncouple respiration in model assay systems, and a chromosomal location to a region with genetic linkage to obesity and hyperinsulinemia led us to initially propose that it was an uncoupling protein involved in the dissipation of excess metabolic fuel as heat. However, a variety of studies on mitochondrial coupling efficiency in isolated mitochondria combined with the absence of obesity and cold sensitivity phenotypes in Ucp2–/– mice has shed doubt on the uncoupling hypothesis (3
4
5
6
7)
. UCP2 protein is present in minute amounts and mostly in tissues with high content of immune cells such as spleen, lung, and intestine (8)
. In these tissues, UCP2 plays a role in both immune and nonimmune cells (9
, 10)
. Studies of Ucp2–/– mice show an increased proinflammatory response in immune cells triggered by an increased reactive oxygen species (ROS) production (5
, 11)
. As a result, Ucp2–/– mice exhibit an enhanced survival to toxoplasmosis (5)
at the cost of an increased susceptibility to pathologies related to oxidative stress such as atherosclerosis and inflammation (9
, 10
, 12)
.
The vast majority of ROS are generated as a byproduct of the mitochondrial respiratory chain activity. It is estimated that at least 0.2% of oxygen consumed by mitochondria is converted to ROS (13)
. Thus, ROS production is tightly correlated to the mitochondrial metabolism of which NADH and acetyl CoA are the central compounds. Cells preferentially use glucose or fatty acid to provide acetyl CoA depending on their energy requirements: proliferative cells use pyruvate as the major substrate for their energetic needs (14)
, whereas quiescent cells catabolize both glycolytic-derived pyruvate and fatty acid. Reactive oxygen species are characterized by their capacity to cause oxidative damage to proteins, DNA, and lipids. The importance of oxidative stress can be appreciated from studies of organisms deficient in antioxidant enzymes and has been associated with aging, carcinogenesis, and various neurodegenerative diseases. In contrast to the nonspecific role of ROS in oxidative stress, growing evidence suggests that ROS may act as second messengers and specifically activate different signaling pathways (15)
. For example, it has been shown that low concentrations of superoxide and hydrogen peroxide stimulate proliferation and enhance cell survival in different cell types (16
17
18)
.
The increased atherosclerosis and decreased susceptibility to certain infections suggest a potential role for UCP2 as a negative regulator of ROS. We, therefore, postulate that UCP2-mediated ROS may also affect cellular proliferation. This study was performed with several cell types using different substrates for energy metabolism. In this work, we demonstrate that Ucp2 knockout modulates cellular glucose sensitivity and as a result cellular proliferation. Consistently with this phenotype, a decrease in fatty acid oxidation along with an increase in glycolysis was observed in the Ucp2–/– cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. MEFs were cultured in DMEM (high glucose, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, penicillin, streptomycin, L-glutamine, and 1 mM nonessential amino acids. They were immortalized by transfecting an SV40 T-antigen plasmid using Lipofectamin (Invitrogen). For glucose withdrawal, cells were washed twice with PBS and then cultured in glucose-free DMEM (Invitrogen) supplemented with 10% FBS, penicillin, streptomycin, L-glutamine, and nonessential amino acids. All experiments with primary MEFs were conducted between passage 2 and 5.
The Chinese hamster ovary (CHO-K1) cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The stable transfected CHO cells overexpressing UCP1 and UCP2 were obtained as described previously in (19)
and (20)
, respectively, and maintained under selection pressure. All CHO cells were cultured in Ham’s F12 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine. Cell proliferation in the absence of glucose or in the presence of etomoxir was performed in the same medium as was used for the MEF experiments.
Measurement of cell proliferation
Early passage primary MEFs (passage<3) were subjected to serial passages according to a modified NIH-3T3 protocol. Cell numbers were determined by cell counting using trypan blue exclusion, and population doublings (PDL) per passage were calculated using the following formula: 
PDL = log2(CF/CI), where CI is the initial number of cells and CF is the number of cells after 3 days of culture. For immortalized MEFs (iMEFs), cells were plated in triplicate at low density (100 cells/well) in 12-well plates from three different 10 cm plates. After 15 days in culture, colonies were exposed to staining solution containing 0.05% crystal violet in 80% ethanol for 30 min. Pictures were taken (x400), or staining was then washed with water and solubilized with ethanol/acetone (80/20%), and the corresponding OD was read at 570 nm. For the T cell proliferation assay, single-cell suspensions from spleen and lymph node were generated by gently pressing the organ through sterile wire mesh. Cells were resuspended in RPMI supplemented with 10% fetal bovine serum, penicillin, streptomycin, and nonessential amino acids. Cells were stimulated for 72 h with the indicated concentrations of CD3 MAb in 96-well U-bottomed plates (Corning Costar, Corning, NY, USA) at a density of 25 x 104 cells per well in 200 µl of RPMI medium supplemented with penicillin, streptomycin, β-mercaptoethanol, and 10% serum. Cells were pulsed with 1 µCi [3H]thymidine per well during the last 24 h. Cells were then harvested and 3H-thymidine uptake was counted. Cell proliferation for CHO cells and proliferation of immortalized MEF in presence of etomoxir and after glucose withdrawal were determined as described previously with iMEFs, except that cells were plated at 1000 cells/well and let grow for 5 days.
Cell migration assay
Studies were performed in Transwell chambers as described by the manufacturer (BD Biosciences, San Jose, CA, USA). For MEFs, cells were serum-starved for 24 h and 5 x 105 cells in serum-free medium were seeded in the upper chamber. Medium with or without serum was used in the lower chamber. After 3 h at 37°C, the cells on the upper surface of the filter were mechanically removed with a cotton swab. The filters were fixed and stained with a crystal violet solution. For T cells, 5 x 105 cells in 300 µl of medium in presence were seeded in the upper chamber; medium with stromal-derived factor-1 (SDF1) was used in the lower chamber. After 3 h at 37°C, the cells in the lower chamber were counted.
Oxygen consumption and ATP/ADP ratio measurements
Oxygen consumption and ATP/ADP ratio were determined as described previously (21)
. For oxygen consumption, cells were trypsinized and resuspended in their medium. Cellular respiration was determined in basal conditions, in the presence of oligomycin (0.5 µg/ml) and finally in the presence of increasing amounts of carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1–20 µM). For ATP/ADP ratio, cells were washed with ice-cold PBS, scraped into perchloric acid, and homogenized. Cell lysates were neutralized with K3PO4, centrifuged at 10,000 g for 20 min, and injected into an HPLC. The area under the curve detected at A254 was used as the quantitative measurements of ATP and ADP, respectively.
ROS production
Superoxide production was measured using the probe dihydroethidium (DHE) as described in (21)
. Briefly, cells were trypsinized and resuspended at 100,000 cells/ml in medium with 1.25 µM DHE supplemented or not with the mitochondrial poisons. The red fluorescence was measured using flow cytometry at different times (0–2 h).
NADH measurements
Primary MEFs were resuspended at 5 x 108 cells/ml in fresh DMEM (high glucose, Invitrogen) supplemented with 10% fetal bovine serum, penicillin, streptomycin, L-glutamine, and 1 mM nonessential amino acids. NAD(P)H fluorescence was measured at an excitation of 340 nm and an emission of 460 nm using a Fluoromax 2 spectrofluorimeter with constant stirring. Since both NADH and NADPH have similar excitation and emission spectra, the fluorescence measured at 340 nm reflected the sum of NADH and NADPH (NAD(P)H). When appropriate, noise editing was use to improve readability of the tracing. The final concentration of CCCP and rotenone used was 5 µM, iodoacetic acid 1µM and KCN 500 µM.
Glycolysis and fatty acid oxidation
The conversion of 3H-glucose to water was used to measure the glycolytic rate as described previously (22)
. Mitochondrial fatty acid oxidation was calculated as the difference between total fatty acid oxidation minus fatty acid oxidation in presence of etomoxir, a CPT1 inhibitor as described in DeBerardinis et al. (23)
.
Western blot analysis
For UCP2 Western blot, mitochondrial protein was isolated from cells, loaded onto a 12% SDS-PAGE gel, transferred onto nitrocellulose, and revealed with the mUCP2–2/3 antibody (8)
. Porin was used as a loading control. For autophagy experiments, whole lysates were prepared and the anti-LC3 antibody was used (24)
. Actin was used as the loading control.
Statistical analysis
Mann-Whitney test was used for statistical analysis. Values are expressed as mean ± SEM. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Interestingly, cells without UCP2 grew faster than cells expressing UCP2 (Fig. 1
A). Nonimmortalized cells undergo a permanent withdrawal from the cell cycle after a discrete number of passages, termed senescence, triggered by DNA damage. Senescence started at the same time in primary MEFs whether or not UCP2 was present (Fig. 1A
, onset of senescence shown by arrow). Reducing oxygen availability from 20 to 3% reduces the flux of electrons through the electron transport chain and, therefore, minimizes ROS production. As a result, MEFs grow faster under 3% oxygen levels (25)
. To determine if the difference observed in cell proliferation was due to an increased resistance of the Ucp2–/– MEFs to oxidative stress, the growth rate for both cell types was determined under 3% oxygen and 21% oxygen (Fig. 1B
). Both wild-type and Ucp2–/– cells grew faster under 3% oxygen. However, the difference between Ucp2–/– cells and Ucp2+/+ cells remained similar (2.2-fold under 3% oxygen vs. 3-fold under 21% oxygen), suggesting that an increased resistance of Ucp2–/– MEFs to oxidative stress cannot explain the difference in proliferation.
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To determine whether UCP2 is associated with proliferation in other cell types, cell proliferation was compared in immortalized MEFs (iMEFs), T cells, and CHO cells overexpressing different amounts of UCP2. MEFs from 14 different embryos of either Ucp2+/+ or Ucp2–/– genotypes were immortalized. The doubling time of iMEFs was four times lower than that of primary MEFs (1.6 vs. 5 days, respectively). Their proliferation was measured by their ability to form colonies when plated at a low density (100 cells/well). In this assay, the number as well as the size of the colonies were increased in Ucp2–/– iMEFs (Fig. 2
A). To rule out any contribution of cell death to the decreased colony formation observed, similar experiments were performed with greater numbers of cells (1000 cells/well). Similarly to the clonogenic assay, the Ucp2–/– cells grew faster than wild-type cells in the presence of 25 mM glucose (see
Fig. 5B
). Since overexpression of UCP2 has been shown to reduce cellular adhesion (26)
, we investigated if the differences in the colony assay could reflect an increased ability of the cells to adhere in the absence of UCP2. iMEFs were starved overnight in 0.1% of serum and then left for 30 min to adhere on plates coated or not with fibronectin. Nonadherent cells were removed, and adherent cells were stained to quantify cellular adhesion. The presence of fibronectin improved cellular adhesion; however, no difference was observed between Ucp2–/– and Ucp2+/+ cells (data not shown). Cell proliferation in T cells isolated either from Ucp2+/+ and Ucp2–/– mice was determined with a thymidine incorporation assay after activation with an anti-CD3 antibody. As observed with MEFs, T cells without UCP2 proliferated to a greater extent than cells expressing the protein (Fig. 2B
).
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Finally, proliferation of wild-type CHO cells and CHO cells overexpressing UCP1 or UCP2 was measured using the colony formation assay. The relative amount of each uncoupling protein overexpressed in CHO cells was determined by Western blot using the mUCP2–2/3 antibody, an antibody raised against UCP2 but recognizing both UCP1 and UCP2 (8)
. Wild-type CHO cells do not express any uncoupling protein (Fig. 2C
, bottom panel). Three stably transfected clones with varying expression levels of UCP2 (S4, S12, and S22, in order of decreasing amount of UCP2 expression) and one clone overexpressing UCP1 at a similar level compared to the clone with the highest UCP2 level (S4) were selected and assayed for their proliferation (Fig. 2C
, bottom panel). CHO cells overexpressing UCP1 exhibited the same proliferation rate as wild-type CHO cells (Fig. 2C
, top panel). The degree of UCP2 overexpression correlated roughly with the inhibition of proliferation, as the highest-expressing CHO cell clone (S4) showed the slowest rate of proliferation, whereas those with intermediate levels of UCP2 expression showed only moderately decreased proliferation (Fig. 2C
). Similar results were obtained when cells were plated at higher density (data not shown). Since cell death could contribute to a lower cell numbers in clonogenic assay, cell proliferation was also measured in standard culture conditions. In these conditions, no cell death was observed, whereas the proliferation was lower in CHO cells overexpressing UCP2 compared to wild-type CHO cells or CHO cells overexpressing UCP1 (data not shown). Altogether, these results show that cell proliferation correlates with the level of UCP2.
Increased proliferation is associated with a metabolic switch driven by increased bioenergetic needs
Cell proliferation can be regulated by different pathways involved in the control of the cell cycle or by the activation of signaling cascades involved for example in nutrient uptake. A cell-cycle analysis showed no difference in the number of cells in each phase of the cell cycle in Ucp2–/– iMEFs compared to Ucp2+/+iMEFs (42±1% vs. 41±2% in G1, 24±1% vs. 24±2% in G2/M and 22±1% vs. 21±2% in S, respectively). This result suggests that in the absence of UCP2 the cell cycle is shortened rather than the number of cells in G1 is decreased. Next we investigated if PI3K/Akt and MAPK signaling pathways, both of which are implicated in cell proliferation, were more activated in Ucp2–/– iMEFs. However, the amounts of both phosphorylated and total levels of these kinases were unchanged in Ucp2+/+ and Ucp2–/– iMEFs (data not shown).
As UCP2 was identified based on its homology to UCP1, known to function as a mitochondrial uncoupler, one might predict that UCP2 knockout would lead to changes in mitochondrial bioenergetics such as mitochondrial respiration, ROS production, or NADH level. To test this hypothesis, mitochondrial ROS production was measured by cytometry analysis using the probe DHE. As expected, addition of rotenone or antimycin, which inhibit the two major sites of mitochondrial ROS production, complex I and complex III, respectively, resulted in a similarly increased level of ROS in both Ucp2+/+ and Ucp2–/– cells (Fig. 3
A). Surprisingly, no significant difference in ROS level was detected between Ucp2+/+ and Ucp2–/– cells. However, the oxygen consumption was significantly increased in the absence of UCP2 both in basal conditions and in the presence of CCCP, a protonophore that dissipates the electrochemical gradient generated by the respiratory chain across the mitochondrial membrane (Fig. 3B
). Similar experiments performed with the DCFDA probe led to similar results, i.e., no difference in ROS production between Ucp2+/+ and Ucp2–/– cells (data not shown). Consistent with these results, Ucp2–/– cells exhibited a decreased ATP/ADP ratio compared to the wild-type (Fig. 3C
). Furthermore, NADH level measured by fluorimetry was significantly lower in Ucp2–/– cells than in Ucp2+/+ cells (22.5±1 in Ucp2–/– cells vs. 24.8±1 in Ucp2+/+ cells, Fig. 3D
). Addition of either rotenone, which inhibits complex I, the major site of mitochondrial NADH consumption, or KCN, which inhibits complex IV, altered the NADH level in a similar manner in Ucp2–/– and Ucp2+/+ cells (Fig. 3D
). No difference was observed in the mitochondrial membrane potential measured by flow cytometry analysis between Ucp2+/+ and Ucp2–/– cells (data not shown) These results suggest that the energy consumption is increased in Ucp2–/– cells, and as a result they respirate faster.
Since cells adapt their metabolism depending on their energy requirements, we set out to determine whether the loss of UCP2 was associated with metabolic changes. Interestingly, the glycolytic rate was significantly increased in the absence of UCP2 (Fig. 4
A), whereas mitochondrial fatty acid oxidation was reduced by two-thirds (Fig. 4B
). This decrease in fatty acid oxidation could explain the lower level of mitochondrial NADH observed in Ucp2–/– cells compared to Ucp2+/+ cells.
Finally, UCP2 expression was determined by Western blot analysis in mitochondria isolated from iMEFs (Fig. 4C
). Even though we could detect the protein in mitochondria from Ucp2+/+ iMEFS, its expression is at least 10 times lower than its level in the spleen, since 50 µg of mitochondria of MEF resulted in a similar or even weaker signal than that of only 5 µg of spleen mitochondria (Fig. 4C
). Altogether, these findings show that the genetic loss of UCP2 induces a metabolic shift toward glycolysis as well as an increased mitochondrial respiration.
Ucp2–/– cells exhibit enhanced dependency to glucose
To explain the metabolic shift occurring in Ucp2–/– cells, we examined the ability of the cells to proliferate in the presence of decreasing availability of essential substrates such as glutamine, glucose, and fatty acid. In the presence of high concentrations of glutamine (2 mM), glucose (25 mM), and fatty acids, Ucp2–/– iMEFs grew significantly faster than wild-type cells (Figs. 2A
and 5)
. Decreasing amounts of glutamine reduced cell proliferation in a similar manner in both Ucp2–/– and Ucp2+/+ cells (Fig. 5
A). Interestingly, whereas Ucp2+/+ cells proliferate at the same rate at 25, 5, or 2 mM glucose, the proliferation rate of Ucp2–/– cells decreased linearly with the amount of glucose present in the media (Fig. 5B
). At 2 mM of glucose, Ucp2–/– cells grew at a similar rate as that of wild-type cells. When glucose was lower than 2 mM, both wild-type and knockout cells proliferated less. No proliferation was observed in the absence of glucose. Fatty acid deprivation was triggered by addition in the medium of etomoxir, an inhibitor of the carrier responsible for fatty acid import into the mitochondria, CPT1. Increasing amounts of etomoxir decreased cell proliferation in both cell types (Fig. 5C
). The proliferation was reduced by nearly 2-fold in the Ucp2+/+ cells and only 1.5-fold in the Ucp2–/– cells. Thus, in contrast to Ucp2+/+ cells, metabolism of Ucp2–/– cells is mostly supported by glucose.
Proliferation of the different CHO cell lines was also measured in the presence of decreasing amounts of glucose and fatty acids. Decreasing glucose from 25 mM to no glucose barely modified cell proliferation in CHO cells overexpressing UCP2, whereas the proliferation of wild-type CHO cells decreased by 2-fold (Fig. 6
A). As in CHO cells, a 1.5-fold decrease in cell proliferation was also observed in CHO cells overexpressing UCP1 when glucose was decreased from 25 to 2 mM (Fig. 6B
). Furthermore, there seemed to be a rough correlation between the level of UCP2 overexpression and the lack of glucose dependence on proliferation, as the highest-expressing UCP2 clone (S4) showed the most constant proliferation rates with decreasing glucose concentrations, whereas intermediate-expressing UCP2 clones (S12 and S22) exhibited only minor decreases in proliferation with decreasing glucose concentrations (Fig. 6B
). Thus, whereas Ucp2-deficient cell lines display increased proliferation and increased sensitivity to decreasing glucose concentration, Ucp2 overexpression leads to the opposite, namely decreased proliferation and a reduced dependence on glucose availability.
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Addition of 100 µM of etomoxir to the cell medium decreased 2-fold the cell proliferation in wild-type CHO cells, whereas no change was observed in CHO cells overexpressing UCP2 (Fig. 6C
). CHO cells overexpressing UCP1 behaved as wild-type CHO cells, with a 2-fold inhibition of cell proliferation in the presence of etomoxir (data not shown). These results suggest that the overexpression of UCP2 in these cells abrogates the inhibition of CPT1 by etomoxir.
To test the hypothesis that cells are more responsive to glucose concentrations in the absence of UCP2, we examined glucose sensitivity of the cells after glucose withdrawal. When eukaryotic cells cannot utilize available metabolic substrate to maintain their ATP/ADP ratio and suppress AMP production, the process of macroautophagy is activated. Macroautophagy activation can be characterized by the conversion of the microtubule-associated protein-1 light chain 3 (LC3) from its native isoform I to a shorter isoform LC3-II (27
28
29)
. In the presence of glucose, cells did not activate this process and LC3 was detected as its unmodified isoform (Fig. 5B
). In contrast, in the absence of glucose, almost half of the LC3 was processed to isoform II in Ucp2–/– cells. Therefore, the absence of UCP2 leads to a hypersensitivity to glucose deprivation. Altogether, these results show that proliferation of the Ucp2–/– cells is highly dependent on glucose availability whereas those of Ucp2+/+ cells relies mostly on fatty acid.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The metabolic shift resulting in increased glycolysis and decreased fatty acid oxidation confers a dependency of the Ucp2–/– cells to glucose since the presence of glucose in the media is not only sufficient but required in Ucp2–/– cells. In contrast, variations of glucose concentration do not alter the proliferation of the Ucp2+/+ cells and overexpression of UCP2 in CHO cells allows the cells to proliferate in the presence of etomoxir at concentrations that normally inhibit proliferation. These results suggest that cells increased glycolysis in order to compensate for the metabolic alteration triggered by the absence of UCP2. Fatty acid oxidation is mostly controlled at the entry of fatty acid into mitochondria. Thus, one can imagine that UCP2 could modulate the entry of fatty acids into the mitochondria. Then, in the absence of UCP2, the decreased fatty acid oxidation limits the production of both acetylCoA and reduced coenzymes. To compensate for the decrease of fatty acid-derived acetylCoA, cells increase import of pyruvate into mitochondria by increasing glycolysis. Interestingly, Ucp2–/– cells exhibit both a decreased NADH level associated with an increased glycolysis, which fits this model. A role for UCPs in lipid metabolism has already been proposed based on the observation that their expression levels are increased during fasting (30
31
32
33
34
35)
. However, no alteration in fatty acid catabolism associated with the presence or the absence of UCP2 has been reported until now. In wild-type cells, the function of UCP2 could be to maintain fatty acid oxidation in cells that otherwise would convert their metabolism to aerobic glycolysis. Considering that UCP2 protein is the most abundant in tissues or cells characterized by a glycolytic metabolism, such as spleen, lung, or immune cells, respectively, such a role could be important in order to provide metabolites such as oxaloacetate necessary for the TCA cycle activity and to deliver reducing equivalents to the respiratory chain.
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It is difficult to distinguish whether the increased proliferation in Ucp2–/– cells is the cause or the result of the observed metabolic modifications. However, it is tempting to postulate that the metabolic shift is the first event to occur in the absence of UCP2, resulting secondarily in increased cell proliferation for several reasons: 1) it is known that the oxidative:glycolytic ratio determines the proliferation rate (36)
; 2) no difference is observed in the cell cycle and in the main signaling pathways controlling proliferation; and 3) the lower ATP/ADP ratio in Ucp2–/– cells is not favorable to increase cellular proliferation since this process requires energy. A glycolytic switch is known to be advantageous for cellular proliferation as long as glucose supplies are not limiting (14)
.
Cell proliferation plays a central role in the activation of the immune system. The proliferative phenotype observed in cells lacking UCP2 could play a role in the alterations of the immune system observed in Ucp2–/– mice (5
, 12)
. It has also been suggested that the glycolytic shift plays a role in tumor progression. Thus, a decreased activity of UCP2 in vivo may be associated with increased tumor development. This hypothesis is supported by some preliminary data showing that Ucp2–/– mice develop more colon tumors than wild-type mice when they are exposed to an alkylating agent (37)
.
The fact that the absence of UCP2 does not increase the ROS production was unexpected since previous studies, including some performed in our lab, showed such results (5
, 11
, 12
, 38
39
40
41)
. Several hypotheses can be advanced to explain this result: 1) the extremely low level of UCP2 in fibroblasts and 2) the lack of specific activators of UCP2 in fibroblasts since uncoupling proteins need to be activated in order to uncouple mitochondrial respiration (6
, 39)
. Both the increased proliferative phenotype of Ucp2–/– cells that persists under conditions of 3% oxygen as well as the lack of difference in cellular senescence, an ROS-dependent process, are consistent with the absence of difference in the ROS production. Altogether these results suggest that the metabolic and proliferative alterations triggered by UCP2 have to be explained by an activity distinct from uncoupling. Although its homology to UCP1 originally led us and others to propose an uncoupling activity for UCP2 in vivo, the present results suggest that UCP2 regulates mitochondrial substrate utilization rather than uncoupling respiratory chain activity from ATP synthesis per se. Identification of the physiological substrate of UCP2 will not be an easy task but is critical for elucidating its biological function.
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ACKNOWLEDGMENTS |
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Received for publication May 4, 2007. Accepted for publication August 2, 2007.
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