Uncoupling Protein 3 (UCP3) Stimulates Glucose Uptake in Muscle Cells through a Phosphoinositide 3-Kinase-dependent Mechanism*

Christine HuppertzDagger §, Britta M. FischerDagger §, Young-Bum KimDagger , Ko KotaniDagger , Antonio Vidal-PuigDagger , Lawrence J. Slieker||, Kyle W. Sloop||, Bradford B. LowellDagger , and Barbara B. KahnDagger **

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 beta -galactosidase was provided by C. Newgard and amplified (27).

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 alpha -MEM with 2% fetal bovine serum or DMEM with 10% fetal calf serum respectively. UCP3 or beta -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.

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 alpha -MEM with 2% fetal bovine serum or in standard alpha -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.

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 (alpha -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 (alpha -subunit) (1:1000) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY).

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 p85alpha 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.

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 alpha -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).

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 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -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 beta -galactosidase basal at p < 0.05.

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 beta -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.

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 beta -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 beta -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 beta -galactosidase; B, cells transduced with UCP3; C, cells transduced with UCP3 grown in media containing 25 mM glucose.

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 beta -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 beta -galactosidase basal at p < 0.05.

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 beta -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 beta -galactosidase basal at p < 0.05.

Fig. 5A shows that UCP3 overexpression increased PI3K activity immunoprecipitated with an antibody against phosphotyrosine 2.2-fold compared with beta -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 beta -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 beta -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.

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 beta -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.

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 beta -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 beta -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.

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.


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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 alpha -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.

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.


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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.

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).


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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 (alpha -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

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 3beta 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.

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.

    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.

    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

    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; alpha -MEM, alpha minimal essential medium.

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