Depletion of Mitochondrial DNA Causes Impaired Glucose Utilization and Insulin Resistance in L6 GLUT4myc Myocytes*

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 μg/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.

The pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM) 1 results from impaired peripheral tissue sensi-tivity to insulin and reduction of insulin secretion. Glucose uptake in mammalian cells is mediated by a family of intrinsic membrane proteins known as facilitative glucose transporters, GLUTs (1,2). GLUT4, the insulin-responsive glucose transporter, is selectively expressed in muscle and adipose cells (3). GLUT4 in these cells constantly recycles between the plasma membrane and intracellular storage pools (4,5). About 90% of GLUT4 is sequestered intracellularly in the absence of insulin or other stimuli such as exercise (6,7). Insulin stimulates glucose uptake in these cells primarily by inducing net translocation of GLUT4 from the intracellular storage sites to the plasma membrane (8,9). The reduction in cellular GLUT4 expression, GLUT4 translocation, and/or impaired insulin signaling would result in insulin resistance and hyperglycemia, the primary characteristics and hallmarks of NIDDM (10).
It is generally accepted that NIDDM is a polygenic disorder composed of subtypes whereby genetic susceptibility is strongly associated with environmental factors (11). Maternally inherited mitochondrial DNA (mtDNA), which encodes 22 tRNAs, 13 subunits of the electron transport system, and its own 16 and 26 S ribosomal RNAs, has been considered one of the genetic factors for the development of diabetes (12,13). Although mtDNA within the cells is heteroplasmic, it has been reported that ϳ0.5-1.5% of diabetics over the age of 40 exhibit mtDNA abnormalities such as duplications (14), point mutations (15), and large scale deletions (16). Furthermore, the cellular oxidative capacity, which is mostly dependent on mitochondrial function, is directly correlated with insulin sensitivity in skeletal muscles (17)(18)(19), and reduced mitochondrial oxidation and phosphorylation determined by NMR are associated with insulin resistance in skeletal muscle of elderly people (20), raising the possibility that impaired mitochondrial function results in insulin resistance. Moreover, the quantitative analysis of mtDNA revealed a decrease in mtDNA content in diabetic animals and humans (21)(22)(23). Similarly cellular content of mtDNA in the pancreatic islets of Goto-Kakizaki rat was markedly decreased without major deletion or restriction fragment polymorphism in mtDNA (21). Southern blot analyses from type 1 and type 2 diabetic patients also revealed that the cellular mtDNA copy number in skeletal muscles was ϳ50% of normal (22). Lee et al. (23) also reported that the quantitative decrease of mtDNA in lymphocytes preceded the type 2 diabetic development, suggesting that the decreased content of mtDNA might be a causal factor in type 2 diabetes. However, there is as yet no convincing evidence that the reduction of cellular mtDNA content in peripheral tissues such as muscle cells is responsible for the disturbances in the glucose uptake and insulin resistance.
It has been reported that long term treatment of cells with low doses (0.1-2 g/ml) of ethidium bromide (EtBr), an inhibitor of DNA/RNA synthesis, specifically suppresses the replication and transcription of extrachromosomal genetic components such as mtDNA without affecting nuclear DNA replication and transcription (24 -26). Therefore, mtDNA-depleted cells have been important tools in the investigation of the cellular components regulated by mtDNA (27). Several lines of evidence obtained from mtDNA-depleted cells have revealed that mitochondrial stress resulting from the reduction of mtDNA causes substantial activation and inactivation of nuclear DNA-encoded gene expression (28 -31). However, it is not clear whether the mtDNA depletion is a contributing factor in the expression of molecules responsible for the insulin signaling and GLUT4 translocation.
In the present study, we report that the depletion of mtDNA in L6 GLUT4myc myocytes is directly correlated with drastic reduction in basal glucose utilization and resistance to insulin stimulation as shown by glucose uptake and GLUT4 translocation. The expression level of IRS-1 was also reduced in the depleted cells with drastic reduction in the insulin-stimulated GLUT4 translocation to the PM, and the insulin-stimulated phosphorylations of IRS-1 and protein kinase B (Akt2/PKB) were decreased in the depleted cells. These findings strongly suggest that the GLUT4 contents in the PM and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content and that the reductions in the expression of IRS-1 and the insulin-stimulated phosphorylation of IRS-1 and Akt2/PKB are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.
Cell Culture-The parent cell line used in this study was L6 GLUT4myc, an L6 cell line (provided by Dr. Amira Klip (the Hospital for Sick Children, Toronto, Canada)) expressing GLUT4-myc, constructed by inserting a human c-myc epitope (14 amino acids) into the first ectodomain of rat GLUT4 (32). Cells were maintained in ␣-minimal essential medium supplemented with 10% fetal bovine serum in a humidified atmosphere of air and 5% CO 2 at 37°C. The L6 GLUT4myc cell line with partially depleted mtDNA was isolated by treating L6 GLUT4myc myocytes with EtBr (0.2 g/ml) and uridine (50 g/ml) for 3 weeks in ␣-minimal essential medium supplemented with 10% fetal bovine serum. The control parental L6 GLUT4myc myocytes were maintained for the same time period in normal culture condition. Since the depletion of mtDNA in myoblast treated with EtBr severely impairs the formation of myotube (33), which makes L6 GLUT4myc myocyte differentiation less quantitative and less reproducible, and since L6 GLUT4myc myocytes without differentiation possess GLUT4 recycling compartments, insulin-sensitive glucose uptake and the insulin signaling intermediates similar to primary muscle cells (34 -39), we used L6 GLUT4myc myocytes to study how insulin stimulates glucose utilization. Myocytes were deprived of serum for 5 h prior to all experimental manipulations.
Staining of Functional Mitochondria-L6 GLUT4myc myocytes were incubated with the mitochondria-specific cationic fluorescent dye, Mitotracker Green (Molecular Probes, Inc., Eugene, OR), for 15 min at 37°C and washed three times with prewarmed phosphate-buffered saline (PBS). The cell preparations were visualized and photographed in a fluorescence microscope (Zeiss). The fluorescent intensity reflects the integrity of mitochondrial function (40). Measurement of Cellular ATP Levels-The cellular ATP was measured by using a somatic cell ATP assay kit (Sigma) as previously described (28). Briefly, the harvested L6 GLUT4myc cells were lysed with "ATP-releasing reagent," and the lysates were assayed with injector-equipped luminometer detection (Lumat LB 9501, Berthold, Germany) for luciferase activity as described in the manufacturer's procedure.
Genomic DNA Extraction and PCR-Total cellular DNA was extracted according to the manufacturer's instructions by using DNeasy Tissue kit (Qiagen, Hilden, Germany). The amplification of mtDNA was performed in a PerkinElmer Life Sciences 2400 PCR thermocycler using the following conditions: 94°C for 2 min (initial denaturation), 94°C for 30 s, 60°C for 30 s, 72°C for 45 s (25 cycles), 72°C for 10 min (final extension). The primers used are shown in Table I.
RNA Preparation-Total RNA was extracted using a standard Trizol RNA isolation protocol (Invitrogen). Briefly, 1 ml of Trizol reagent was mixed with cell pellet (5 ϫ 10 5 cells) by repeated pipetting. DNA and protein were excluded by chloroform phase separation. RNA in the aqueous phase was precipitated with isopropyl alcohol and then resuspended in diethyl pyrocarbonate-treated water. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm.
RT-PCR Analysis-RT-PCR analysis was performed as described previously (41). The PCR conditions were established in our laboratory to allow comparisons between the expression of transcripts, which relate insulin signaling and glucose uptake. Under these conditions, the efficiency of the RT-PCR for each gene did not plateau, and the number of cycles used in these experiments was kept to a minimum. The relative expression levels of mRNA were determined by using 1.0 g of total RNA as template and the optimal cycles of PCR with the optimal thermal conditions ( Table I). As a control, we analyzed the transcription levels of ␤-actin and glyceraldehyde-3-phosphate dehydrogenase using specific rat primers, the same amount of total RNA, and 25 PCR cycles. These were optimal conditions for the analysis of mRNA expression, and the analysis was limited to the products generated in the exponential phase of the amplification. After RT-PCR, the content of each independent reaction tube was subjected to electrophoresis on 2-3% agarose gels containing EtBr. Images from electrophoresed gels were captured by a camera in a computer-assisted imaging system (AlphaImager 1220; Alphainotech Co., San Leandro, CA).
Quantitative Gene Expression Analysis-In order to confirm gene expression levels, quantitative real time RT-PCR (qRT-PCR) was carried out in Rotor Gene 2000 (Corbett Research, Mortlake, Australia) using SYBR-Green PCR Master Mix according to the manufacturer's instructions (Qiagen, Valencia, CA). All of the gene-specific primer sets used for qRT-PCR are listed in Table I. After amplification, the authenticity of the PCR products was verified by melting curve analysis and agarose gel electrophoresis. Images from electrophoresed gels were captured by a camera in a computer-assisted imaging system (AlphaImager 1220). The qRT-PCR results were analyzed using Rotor-Gene analysis software version 6.0 (Corbett Research). The comparative cycle threshold (C T ) method was used to analyze the data by generating relative values of the amount of target cDNA. Relative quantitation for any given gene, expressed as percentage variation over control, was calculated after determination of the difference between C T of the given gene A and that of the calibrator gene B (␤-actin) in the depleted or reverted myocytes (⌬C T1 ϭ C T1A Ϫ C TB ) and control myocytes (⌬C T0 ϭ C T0A Ϫ C TB ) using the 2 Ϫ⌬⌬CT(1-0) formula. C T values are means of triplicate measurements. Experiments were repeated 3-5 times.
Determination of 2-Deoxyglucose (2DG) and 3-O-Methylglucose (3OMG) Uptake-Cells were starved of serum for 4 h and washed twice with Hepes-buffered saline solution (140 mM NaCl, 20 mM Na-Hepes, 2.5 mM MgSO 4 , 1 mM CaCl 2 , and 5 mM KCl, pH 7.4). 2DG and 3OMG uptakes were measured as described previously (42). Briefly, cells were incubated for 20 min in the presence or absence of insulin (100 nM) and then 2DG uptake (10 M [ 14 C]2DG, 1 Ci/ml, PerkinElmer Life Sciences) was measured for 3 min. Nonspecific uptake was determined in the presence of 10 M cytochalasin B and was subtracted from all values. 3OMG uptake was measured in a similar fashion with the following differences: 50 M [ 14 C]3OMG (4 Ci/ml; PerkinElmer Life Sciences) was added to Hepes-buffered saline solution, and uptake was allowed to occur for 15 s, a period over which 3OMG uptake is known to be linear. Subcellular Fractionation-Membrane preparations were performed as described previously for wild type L6 cells (34). Equal amounts of membrane proteins were analyzed by SDS-PAGE and electrotransferred onto nitrocellulose paper filters.
Gel Electrophoresis and Immunoblotting-Membranes solubilized in Laemmli solution were subjected to SDS-PAGE on 8 or 10% resolving gels as described (43). Separated proteins were electrophoretically transferred to nitrocellulose membrane (Bio-Rad), blocked with 5% nonfat milk in Tris-buffered saline, and incubated with primary antibodies in TTBS (Tris-buffered saline plus 0.05% Tween 20) containing 1% nonfat milk. After overnight incubation, membranes were washed with TTBS and incubated with horseradish peroxidase-labeled protein A (for the detection of polyclonal antibodies) or horseradish peroxidaselabeled anti-mouse IgG (for the detection of monoclonal antibodies). Proteins were visualized using an enhanced chemiluminescent substrate kit (PerkinElmer Life Sciences). Immunoblot intensities were quantitated by densitometry using an analytical scanning system (AlphaImager 1220).
Measurement of GLUT4myc Translocation-The movement of myctagged GLUT4 to the cell surface was measured by an antibody-coupled colorimetric assay (34) with slight modifications. Quiescent L6 GLUT4myc cells treated as indicated were washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 3 min at room temperature, and the fixative was immediately neutralized by incubation with 1% glycine in PBS at 4°C for 10 min. The cells were blocked with 3% bovine serum albumin in PBS at room temperature for 1 h. Primary antibody (anti-c-myc, 9E10) was then added into the cultures at a dilution of 1:100 and maintained for 30 min at 4°C. The cells were extensively washed with PBS before introducing peroxidase-conjugated rabbit anti-mouse IgG (1:1000). After 30 min at 4°C, the cells were extensively washed, and 1 ml of O-phenylenediamine dihydrochloride reagent (0.4 mg/ml O-phenylenediamine dihydrochloride and 0.4 mg/ml urea hydrogen peroxide in 0.05 M phosphate/citrate) was added to each well for 10 min at room temperature. The reaction was stopped by the addition of 0.25 ml of 3 N HCl. The supernatant was collected and the optical absorbance was measured at 492 nm. For phosphoprotein analysis, total cell lysates were prepared as described previously with modifications (44). Following appropriate incubation in the presence or absence of insulin (100 nM), cells were lysed on ice with 150 M 2ϫ Laemmli sample buffer supplemented with 7.5% ␤-mercaptoethanol, phosphatase inhibitors (1 mM Na 2 VO 4 and 100 nM okadaic acid), and protease inhibitors (1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 10 M E-64, 1 M pepstatin A, and 1 M leupeptin), passed five times through a 24-gauge syringe, and heated for 15 min at 65°C. Total protein (50 g) was resolved by SDS-PAGE and immunoblotted with the respective phosphospecific antibody.
Statistical Analysis-Values are expressed as the mean Ϯ S.E. Where applicable, the significance of difference was analyzed using Student's t test for unpaired data.

RESULTS
Long term treatment with low doses of EtBr is known to selectively inhibit mtDNA replication and transcription without detectable changes in nuclear DNA (24 -26). In a recent study, mtDNA of the C2C12 muscle cell line was partially depleted by long term exposure to EtBr, and the reversal of mtDNA depletion was successfully achieved by removal of EtBr (28). The ability to partially deplete and replete the cellular mtDNA levels in cell lines by EtBr treatment would allow one to study cellular processes affected by changes in mtDNA content. To develop partially mtDNA-depleted muscle cell lines, we exposed L6 GLUT4myc myocytes to EtBr (0.2 g/ml) in Dulbecco's modified Eagle's medium supplemented with pyruvate and uridine, previously shown to be essential for the growth of mtDNA-depleted cells (27). The mtDNA content from L6 GLUT4myc myocytes cultured with or without EtBr was monitored routinely by amplifying genomic DNA as described under "Experimental Procedures." Fig. 1, A and B, shows that cytochrome oxidase subunits I (COX I) and II (COX II), which are encoded only in mtDNA, were hardly amplified from the genomic DNA of the cells treated with EtBr for 3 weeks. In contrast, nuclear DNA-encoded controls such as COX IV (Fig.  1) and ␤-actin (data not shown) were equally detected in both control and EtBr-treated cells, indicating that prolonged treatment with EtBr partially depleted mtDNA without altering the nuclear DNA replication in L6 GLUT4myc myocytes. Under these experimental conditions, mtDNA was depleted to Ͻ10% of normal cells after 3 weeks of growth in the presence of EtBr (Fig. 1A). In addition, the removal of EtBr from medium successfully normalized mtDNA content (Ͼ90% of the normal cells) in this clone within 7 days (Fig. 1, A and B). In order to minimize the variability of mtDNA content between each experiment, we selected and propagated the single cell clone, whose mtDNA content was less than 5% of the control, and used cells from the same frozen stock for all of the experiments.
In previous reports, the partial depletion of mtDNA reduced mitochondrial transmembrane potential, ⌬⌿ m , as measured with the mitochondria-specific cationic fluorescent dye Mitotracker (28,30,45,46). Since Mitotracker localizes in mitochondria in a ⌬⌿ m -dependent manner (40), the fluorescent intensity from living cells thoroughly reflects the integrity of mitochondrial function. As shown in Fig. 2A, control cells exhibited granule-like fluorescence dispersed in the cytoplasm, whereas the mtDNA-depleted cells showed drastically reduced fluorescence intensity, suggesting that the depletion of mtDNA disrupted ⌬⌿ m in L6 GLUT4myc myocytes. On the other hand, the reverted cells nearly regained the fluorescence intensity of control cells, indicating that mitochondrial membrane potential was nearly restored in the reverted cells. As expected from the depletion of mtDNA content and mitochondrial membrane potential, the depleted cells showed drastically reduced total cellular ATP as compared with control cells (Fig. 2B). Following mtDNA repletion, total cellular ATP content of the reverted cells was not significantly different from that of control cells (Fig. 2B).
We next analyzed the differential expressions of mitochondrial and nuclear genes such as COX I, II, and IV by qRT-PCR in the control, mtDNA-depleted, and mtDNA-reverted cells (Fig. 3, A and B). ␤-Actin used as a control was not changed by mtDNA content alteration by RT-PCR (data not shown). The mtDNA-depleted cells contained very small amounts of COX I and II mRNAs, which are mtDNA-encoded genes, whereas the reverted cells showed a similar amount of COX I and II mRNAs mtDNA-depleted, and mtDNA-reverted L6 GLUT4myc myocytes. A, control, mtDNA-depleted, and mtDNA-reverted L6 GLUT4myc myocytes were stained with MitoTracker Green, a mitochondrial membrane potential dye, as described under "Experimental Procedures." Magnification is ϳϫ400. B, total cellular ATP levels were measured by the luciferin-luciferase assay as described under "Experimental Procedures." ATP content was expressed in arbitrary units, where the ATP content from control L6 GLUT4myc myocytes was set to 100%. Data were analyzed by Student's t test. All results represent mean Ϯ S.E. from five independent experiments. Open column, control; closed column, mtDNA-depleted; gray column, mtDNAreverted; ***, p Ͻ 0.001. FIG. 1. mtDNA content. A, genomic DNA was isolated from control (C), mtDNA-depleted (D), and mtDNA-reverted (R) L6 GLUT4myc myocytes, and mtDNA-encoded genes, such as COX I and COX II, and nuclear DNA-encoded gene, such as COX IV, were amplified by PCR. The PCR products were visualized on agarose gel after EtBr staining. B, the DNA levels obtained by densitometry were normalized against ␤-actin signals (data not shown), and relative intensities were expressed in arbitrary units where the intensity of control L6 GLUT4myc myocytes was set to 100%. Data were analyzed by Student's t test. All results represent mean Ϯ S.E. from eight independent experiments. Open column, control; closed column, mtDNA-depleted; gray column, mtDNA-reverted; ***, p Ͻ 0.001. as control cells. However, mRNA content of COX IV, which are encoded by nuclear DNA, were not changed in the depleted and reverted cells, suggesting that mitochondrial mRNA levels correspond to the cellular mtDNA contents in these cells.
In previous reports, the quantitative analysis of mtDNA revealed that the decrease of mtDNA content is associated with diabetes in animals and human subjects (21)(22)(23). To demonstrate how the depletion and repletion of mtDNA content in L6 GLUT4myc myocytes correlate with glucose transport activity, we measured 2DG uptake in the control, mtDNA-depleted, and mtDNA-reverted cells (Fig. 4A). Insulin stimulation (100 nM, 20 min at 37°C) increased 2DG uptake by 1.95-fold as compared with the basal state in control cells, whereas there was a significant (62%) reduction in the basal state 2DG uptake from the mtDNA-depleted cells. Moreover, insulin-stimulated increase in 2DG uptake was not observed in these cells, indicating that mtDNA depletion in L6 GLUT4myc myocytes provoked impaired glucose utilization and insulin resistance. Interestingly, when the mtDNA content was returned to nearly control level in the reverted cells, 2DG uptake in the presence or absence of insulin stimulation was dramatically restored to that of the control. To confirm that the reduced 2DG uptake in mtDNA-depleted cells is indeed due to a reduced trans-membrane glucose transport rather than a decreased glucose phosphorylation due to the reduced ATP level, we also measured the uptake of nonmetabolizable glucose analogue, 3OMG (Fig. 4B), and obtained results similar to those of 2DG uptake, clearly indicating that the glucose transport step was affected by the reduction of mtDNA in L6 GLUT4myc myocytes.
It has been reported that basal and insulin-stimulated glucose uptake in insulin sensitive tissues, such as muscle and adipocytes, mainly correlates with the amount of GLUT4 in the PM (10,47). Since basal glucose uptake was significantly decreased by mtDNA depletion (Fig. 4), we next assessed whether the reduction of mtDNA content in L6 GLUT4myc myocytes is associated with impaired GLUT4 expression. As shown in Fig.  5A, total cellular GLUT4 contents were not affected by mtDNA alteration and not significantly different among the control, depleted, and reverted cells. However, PM GLUT4 in the basal state of mtDNA-depleted cells was decreased by 40 -60% of control level, whereas repletion of mtDNA normalized PM GLUT4 content (Fig. 5, A and B), indicating that mtDNA depletion may interfere with basal GLUT4 targeting or recycling in L6 GLUT4myc myocytes. It is most likely that the reduced basal 2DG uptake observed in the depleted cells may result from the reduction of PM GLUT4 level as compared with control and reverted cells. Insulin stimulation substantially (about 2-fold) increased GLUT4 translocation to the PM in control cells (Fig. 5, A and B). In the depleted cells, however, with the accompanying reduction in PM GLUT4 content, insulin-stimulated GLUT4 translocation was largely (more than 80%) obliterated (Fig. 5, A and B), indicating that depletion of the cellular mtDNA is closely associated with the development of insulin resistance in L6 GLUT4myc myocytes. The insulinstimulated GLUT4 translocation in the reverted cells was slightly lower than that of control cells, a change that was not statistically significant (Fig. 5B).
Since the expression of various nuclear DNA-encoded genes was affected by mtDNA depletion (28,30,45,46), it is possible that the insulin resistance in the mtDNA-depleted cells observed above may result from alteration in the insulin signaling intermediates involved in GLUT4 translocation, including insulin receptor, IRS-1, PI3K, PDK-1, and their downstream effector, Akt2/PKB (48,49). We measured the steady-state levels of mRNA (Fig. 6, A and B) and protein of these insulin signaling intermediates (Fig. 6, C and D) by qRT-PCR and immunoblotting. Interestingly, the mRNA level of IRS-1 was markedly altered by the depletion in cellular mtDNA content, whereas the mRNA levels of insulin receptor, PI3K, PDK-1, and Akt2/PKB were not affected by the depletion and repletion of mtDNA (Fig. 6, A and B). In the depleted cells, mRNA of IRS-1 was reduced by 76%, compared with control cells. A prominent and selective change in IRS-1 was also evident in the protein levels from the depleted cells (Fig. 6, C and D); the expression level of IRS-1 was reduced by 90% in the mtDNAdepleted cells as compared with control cells. In contrast, protein expression of IRS-2, PI3K, PDK-1, and Akt2/PKB were not changed by mtDNA depletion and repletion. The protein level of IRS-1 was not fully increased to the level of control cells in the reverted cells, the significance of which is not immediately clear. We also checked whether the mRNA levels of several proteins thought to be involved in insulin-stimulated GLUT4 translocation are affected by mtDNA content changes. As shown in Fig. 7, the transcription levels of syntaxin 4, SNAP23, VAMP 2, and TC10 were not significantly different between the control, mtDNA-depleted, and mtDNA-reverted L6 GLUT4myc myocytes. These results suggest that the expression of IRS-1, which is the insulin signaling molecule encoded by nuclear DNA, is selectively affected by the amount of cellular mtDNA in L6 GLUT4myc myocytes, and this change is most likely associated with insulin resistance developed in mtDNA-depleted cells.
We also assessed the effect of mtDNA depletion on the insulinstimulated phosphorylation of IRS-1 and Akt2/PKB in these cells. Cellular insulin treatment drastically increased phosphorylation of IRS-1 as compared with the basal level (Fig. 8). However, mtDNA depletion vastly affected the expression and phosphorylation levels of IRS-1 (Fig. 8). The expression of IRS-1 measured in the depleted cells was decreased by ϳ80% of the control level, and the insulin-stimulated phosphorylation of IRS-1 nearly disappeared. Upon mtDNA repletion, insulin-stimulated phosphorylation of IRS-1 returned to near control. In similar experiments, insulin-stimulated phosphorylation of Akt2/ PKB was also drastically increased in the control and reverted cells, whereas it was nearly totally abolished by mtDNA depletion. It is therefore suggested that the reduction of IRS-1 expression and insulin-stimulated phosphorylation of IRS-1 and Akt2/ PKB result in impaired insulin-stimulated GLUT4 translocation and glucose uptake in mtDNA-depleted cells.

DISCUSSION
Reduction of oxidative enzyme activities impairs regulation of glucose and lipid metabolism in skeletal muscle and is closely associated with the development of insulin resistance in obesity and NIDDM (50,51). Since the bioenergetic capacity of skeletal muscle mitochondria is impaired in NIDDM and obesity (52), prolonged disturbances in the function of mitochondria may be involved in the development of insulin resistance in skeletal muscle. Accumulation of monogenic abnormalities in mtDNA (14 -16) and reduction of mtDNA content are also described in diabetic animals and human subjects (21)(22)(23), suggesting that the decrease of mtDNA content might be a causal factor in type 2 diabetes. However, there is as yet no convincing evidence indicating whether the reduction of cellular mtDNA content in peripheral tissues, such as muscle cells, causes substantial disturbances in glucose uptake and insulin signaling with the development of insulin resistance.
To investigate the correlation between the alteration of mtDNA content and insulin resistance, here we used insulinsensitive L6 myocytes stably expressing an extracellular myc epitope-tagged GLUT4 (L6 GLUT4myc) (34,35). GLUT4 stably Cells were incubated for 20 min in the presence or absence of insulin (100 nM at 37°C) and washed twice with cold Hepes-buffered saline solution. A, total and PM proteins were prepared as described under "Experimental Procedures." 2 g of membrane protein were analyzed by SDS-PAGE and immunoblotting. The protein levels obtained by densitometry from immunoblot intensity were expressed in arbitrary units, where the intensity for control was set to 1. B, the movement of myc-tagged GLUT4 to the cell surface was measured by an antibody-coupled colorimetric assay as described under "Experimental Procedures." Values are expressed as mean Ϯ S.E. from three independent experiments. Open column, basal state; closed column, insulinstimulated state; ***, p Ͻ 0.001. expressed in L6 GLUT4myc myocytes constantly recycles between the PM intracellular storage pools without differentiation or myotube formation (35,36). Approximately 90% of GLUT4 in this cell is sequestered in intracellular endosomes in the absence of insulin stimulation (36). Insulin stimulates glucose uptake in this cell by inducing net translocation of GLUT4 from the intracellular storage sites to the PM (36). Moreover, insulin-stimulated glucose uptake is completely blocked by wortmannin and the cytoskeleton disruptors (34), and insulin-dependent stimulation of PI3K or Akt activities was observed as in primary muscle and adipocytes (34,35). The translocation of GLUT4 from intracellular storage to the plasma membrane was decreased by expression of dominant negative mutants of PI3K and Akt2/PKB (37) and was sensitive to agents that prevent actin remodeling (38,39). Thus, L6 GLUT4myc myocytes represent an appropriate cellular model to study how insulin stimulates glucose utilization.
The cellular treatment with low dose EtBr specifically inhib-FIG. 6. Effect of mtDNA content changes on insulin signaling pathway intermediates. A, total RNA from control, mtDNA-depleted (Depleted), and mtDNA-reverted (Reverted) L6 GLUT4myc myocytes was prepared as described under "Experimental Procedures." Insulin receptor, IRS-1, PI3K, PDK-1, and Akt2/PKB mRNA levels were analyzed by RT-PCR and visualized on agarose gel. B, in order to confirm transcript levels, qRT-PCR was carried out as described under "Experimental Procedures." The comparative C T method was used to analyze the data by generating relative values of the amount of target cDNA. The relative values were expressed in arbitrary units, where the intensity of control L6 GLUT4myc myocytes was set to 100%. C, a representative immunoblot of IRS-1, IRS-2, PI3K, PDK-1, Akt2/PKB, and ␤-actin protein in control, mtDNA-depleted (Depleted), and mtDNA-reverted (Reverted) L6 GLUT4myc myocytes. Total cell lysates (50 g) from each sample were subjected to immunoblot analysis using specific antibodies. D, the protein levels obtained by densitometry in control (C), mtDNA-depleted (D), and mtDNA-reverted (R) L6 GLUT4myc myocytes were normalized against ␤-actin signals, and relative intensities were expressed in arbitrary units, where the intensity for control was set to 100%. Values are expressed as mean Ϯ S.E. from eight independent experiments. Open column, control; closed column, mtDNA-depleted; gray column, mtDNA-reverted; ***, p Ͻ 0.001 versus control.
its transcription and replication of circular DNA by deleting RNA primers required for the initiation of replication (24 -26) and has been a useful tool for the study of cellular responses affected by changes in mtDNA contents (28 -30). In the present study, we generated a set of L6 GLUT4myc cell lines containing partially depleted mtDNA (Ͻ95% of control level) by EtBr treatment, followed by single cell cloning. This mtDNA depletion and repletion protocol has been successfully applied to C2C12 cells (28 -30) and HeLa cells (53), although the concentration and duration of EtBr treatment in each cell type having different susceptibility to EtBr were modified. Partially mtDNA-depleted L6 GLUT4myc myocytes exhibited normal nuclear DNA content, disruption of mitochondria ⌬⌿ m , and significantly reduced cellular ATP content (Figs. 1 and 2). These changes were reversed to nearly normal levels in the reverted cells, whose mtDNA content recovered upon retrieving EtBr from the culture medium ( Figs. 1 and 2). Together, these findings indicate that the depletion and repletion protocol applied here to L6 GLUT4myc myocytes effectively altered mtDNA content without causing general cell damage and is appropriate for studying the effect of mtDNA alteration on glucose utilization and insulin sensitivity.
It is noteworthy that the depletion of mtDNA significantly reduced glucose uptake in basal and insulin-stimulated L6 GLUT4myc cells, whereas the repletion of mtDNA corrected these changes to nearly control levels (Fig. 4). Both 3OMG uptake and 2DG accumulation assays revealed a similar reduction in glucose uptake, validating the 2DG accumulation as a genuine transport assay free of ATP depletion artifacts. In previous studies, Antonetti et al. (22) found that skeletal muscle from type 1 and type 2 diabetic patients contains relatively less mtDNA than normal, and Lee et al. (23) also reported that decreased peripheral mtDNA content occurs before the onset of diabetes. More recently, it has been demonstrated that basal and insulin-stimulated glucose uptake was significantly reduced in the insulin-insensitive SK-Hep1 hepatocyte upon mtDNA depletion by EtBr treatment (46). Our demonstration that basal glucose uptake in L6 GLUT4myc myocytes was modulated by the depletion and repletion of mtDNA (Fig. 4) is consistent with these findings and further supports the possibility that mtDNA-depleted myocytes develop severe insulin resistance.
Although many of the acute effects of insulin on glucose transport are mediated by the recruitment of GLUT4 to the PM, decreased expression of GLUT4 can cause insulin resistance (54), and increased expression of GLUT4 promotes insulin-stimulated glucose uptake (55). In L6 GLUT4myc myocytes stably expressing GLUT4, the total cellular GLUT4 amount was not affected by the changes of mtDNA content (Fig. 5A), whereas GLUT4 in the PM was clearly decreased by mtDNA depletion (Fig. 5A). Since basal and insulin-stimulated glucose uptake in muscle and adipose cells largely correlates with the absolute amount of GLUT4 in the PM, the reduced basal 2DG uptake observed in the depleted cells most likely results from the reduction of PM GLUT4 level.
How at the molecular and cellular levels mtDNA depletion causes a reduction in PM GLUT4 content and insulin resistance is an important question yet to be answered. Our demon- FIG. 7. mRNA content of vesicle trafficking intermediates in control, mtDNA-depleted, and mtDNA-reverted L6 GLUT4myc myocytes. A, total RNA from control, mtDNA-depleted (Depleted), and mtDNA-reverted (Reverted) L6 GLUT4myc myocytes was prepared as described under "Experimental Procedures." Syntaxin 4, SNAP23, VAMP2, TC10, and ␤-actin mRNA levels were analyzed by RT-PCR and visualized on agarose gel. B, in order to confirm transcript levels, syntaxin 4, SNAP23, VAMP2, and TC10 mRNA were analyzed by qRT-PCR using the specific primers indicated in Table I. The comparative C T method was used to analyze the data by generating relative values of the amount of target cDNA. The relative values were expressed in arbitrary units, where the intensity of control L6 GLUT4myc myocytes was set to 100%.
FIG. 8. Effect of mtDNA content changes on the expression and phosphorylation of IRS-1 and Akt2/PKB. Control, mtDNA-depleted (Depleted), and mtDNA-reverted (Reverted) L6 GLUT4myc myocytes were starved of serum for 5 h. Cells were preincubated in the absence or presence of insulin (100 nM for 20 min), and total cell lysates were prepared as described under "Experimental Procedures." Total cell lysates (50 g of protein) were subjected to 8% SDS-PAGE, and the expression (IRS-1 and Akt2/PKB) and phosphorylation (pIRS-1 and pAkt2/PKB) levels were determined by immunoblotting with specific antibodies described under "Experimental Procedures." stration that the mtDNA depletion causes a drastic reduction in the expression of IRS-1 and its insulin-stimulated phosphorylation (Figs. 6 and 8) clearly suggests that IRS-1 plays a key role in this process. The effect of mtDNA depletion on IRS-1 expression is quite selective. The mtDNA depletion did not affect the expression of other insulin signaling intermediates such as insulin receptor, PI3K, PDK-1, and Akt2/PKB (Fig. 6).
Several key proteins essential to vesicle trafficking/exocytosis, including syntaxin 4, SNAP23, VAMP2, and TC10 were also not affected by the mtDNA depletion (Fig. 7), indicating that the regulation occurs within the insulin signal transduction prior to GLUT4 vesicle trafficking. Alterations in mtDNA quantity and quality have been known to induce mitochondriato-nucleus stress signaling and change the expression level of various nuclear DNA-encoded genes in vertebrate cells (28 -31). Insulin regulates GLUT4 translocation largely through the activation of IRS-1 via tyrosine phosphorylation with subsequent activation/phosphorylation of its downstream effectors, viz. PI3K, PDK-1, protein kinase C /, and Akt2/PKB (56,57). Although we find that insulin-stimulated Akt2/PKB phosphorylation is also reduced after mtDNA depletion (Fig. 8), it is possible that the mtDNA depletion reduces tyrosine phosphorylation of IRS-1 selectively, and the reduced insulin-stimulated Akt2/PKB phosphorylation observed in mtDNA-depleted cells is mostly due to the reduced IRS-1 phosphorylation. IRS-1 is suggested as one of the critical mediators for the insulinstimulated GLUT4 translocation and glucose uptake in skeletal muscle. The reduced expression of IRS-1 and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle has been observed in both type 2 diabetes (58, 59) and animal models (60 -62). Studies using IRS-1 or -2 knock-out mice also suggested that IRS-1 plays a major role in insulin-stimulated glucose transport, and IRS-2 may only partially compensate for defects in IRS-1 signaling (63,64).
How does mtDNA depletion decrease the amount and phosphorylation of IRS-1 at the molecular level? It is tempting to suggest a role for protein kinase C and ceramides, two key intermediates of lipotoxicity produced by decreased fatty acid oxidation associated with mitochondrial dysfunction, in this process. Free fatty acids and cellular stress can result in increased serine phosphorylation and reduced tyrosine phosphorylation of IRS-1, thereby attenuating insulin signaling and causing insulin resistance in type 2 diabetes (65,66). Protein kinase C phosphorylates IRS-1 at serine 1101, blocking IRS-1 tyrosine phosphorylation and downstream activation of the Akt2/PKB (67). Undoubtedly, more work is needed to determine the molecular events underlying the modulation of IRS-1 expression and its insulin-stimulated phosphorylation by cellular mtDNA content change.
In conclusion, our data clearly show that the depletion of mtDNA is directly correlated with a drastic reduction in basal and insulin-stimulated glucose uptake and GLUT4 translocation in L6 GLUT4myc myocytes. It is also clear in our data that the depletion of mtDNA causes a selective and reversible reduction in IRS-1 mRNA and protein levels and in insulinstimulated IRS-1 and Akt2/PKB phosphorylation. We suggest that depletion of mtDNA causes insulin resistance in L6 GLUT4myc myocytes primarily by reducing the mRNA and protein expression and the insulin-stimulated phosphorylation of IRS-1, a key intermediate of the insulin signal pathway leading to insulin-stimulated GLUT4 recruitment to the PM.