Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Kyu-Sang Park1, Kyung-Jay Nam1, Jun-Woo Kim1, Youn-Bok Lee1, Chang-Yeop Han1, June-Key Jeong2, Hong-Kyu Lee1,3, and Youngmi Kim Pak1

1 Division of Metabolic Disease, Department of Biomedical Sciences, National Institute of Health, Seoul 122-701; and Departments of 2 Nuclear Medicine and 3 Internal Medicine, School of Medicine, Seoul National University, Seoul 110-744, Korea


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho 0 SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho 0 cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho 0 cells compared with parental rho + cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.

oxidative phosphorylation; glucose uptake


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIABETES MELLITUS (DM) is a genetically heterogeneous disorder but exhibits common features of glucose intolerance and hyperglycemia (25). It is generally considered that various genetic factors, although not all are known, cause DM. Maternally inherited mitochondrial DNA (mtDNA), which does not follow the classical Mendelian genetics, has been considered to be one of the genetic factors for developing diabetes (14).

Approximately 0.5-1.5% of all diabetic patients exhibit pathogenic mtDNA mutations such as duplications (2), point mutations (18), and large-scale deletions (3). Also, diabetics show not only mutations but also quantitative changes of mtDNA. Antonetti et al. (1) reported that mtDNA copy number was ~50% decreased in skeletal muscles of both type 1 and type 2 diabetic patients as estimated by Southern blot analyses. Lee et al. (22) 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 for type 2 diabetes. However, there is as yet no convincing evidence whether the reduction of mtDNA copy number causes enough disturbance in the glucose metabolism in peripheral tissues such as liver cells to participate in the development of diabetes.

In pancreatic beta -cells, glucose-induced ATP production stimulates insulin secretion via mitochondrial oxidative phosphorylation. The depletion of mtDNA impairs this glucose-stimulated insulin secretion and causes glucose intolerance (16, 31). The impairment of glucose uptake via glucose transporter in peripheral tissues may also contribute to the diabetic pathogenesis, especially of type 2 (5). For example, population studies have suggested that genetic variation in the GLUT-1 gene, an isoform of glucose transporter, is associated with an increased risk for developing type 2 diabetes (23). To our knowledge, however, there has been no study showing that the alteration of mtDNA copy number affects glucose utilization and metabolism in peripheral tissues.

In this study, we established mtDNA-depleted (rho 0) human hepatoma SK-Hep1 cells and investigated the effects of mtDNA depletion on glucose metabolism to test whether the decrease in mtDNA could participate in the diabetic pathogenesis. The rho 0 cells, established by long-term treatment with ethidium bromide (EtBr), have been important tools in investigating the function of mtDNA (21). EtBr intercalates into circular DNA and inhibits mtDNA replication and transcription at a low concentration (0.1-2 µg/ml) without any detectable effect on nuclear DNA division (16, 35). We succeeded in isolating the rho 0 human hepatoma cell line, which lost the ability of oxidative phosphorylation. We observed that cellular ATP production and glucose uptake were attenuated in rho 0 cells. Glucose transporter expression and glucose metabolizing enzyme activities in rho 0 cells were also lower than in control cells. These results suggest that the decrease in mtDNA copy number may suppress glucose uptake and metabolism, which may lead to the development of glucose intolerance and diabetes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and cell culture. Cells from the human hepatoma cell line SK-Hep1 (American Type Culture Collection, HTB-52, Rockville, MD) were grown in DMEM containing 1 mM pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 µg/ml). They were incubated at ambient oxygen concentrations in the presence of 5% CO2 at 37°C. The mtDNA-depleted (rho 0) cell line was isolated by treating SK-Hep1 cells with 0.1 µg/ml EtBr and 50 µg/ml uridine for >10 wk in DMEM supplemented with 10% FBS. The control parental SK-Hep1 cells were maintained for the same time period in normal culture medium.

PCR and RT-PCR analyses. To compare the mtDNA content between control and rho 0 SK-Hep1 cells, total cellular DNA was extracted using QIAmp Tissue Kit (Qiagen, Hilden, Germany). The PCR amplification of human mtDNA was performed in a volume of 50 µl. This contained 0.25 units of Taq polymerase (Perkin-Elmer, Norwalk, CT) and 10 pmol of oligonucleotide primer pair, which was sequence specific for human mtDNA: upstream 5'-CCT AGG GAT AAC AGC GCA AT-3' and downstream 5'-TAG AAG AGC GAT GGT GAG AG-3' (630-bp product). The PCR conditions for mtDNA included an initial denaturing for 5 min and then 30 cycles as follows: denaturing for 30 s at 94°C, annealing for 30 s at 60°C, and extending for 40 s at 72°C using GeneAmp (Perkin-Elmer). Aliquots of the PCR reactions were analyzed by 1.1% agarose gel electrophoresis and were examined for the size-predicted products by EtBr staining.

For quantification of mRNA expression, RT-PCR was performed. Total RNA from the cells was prepared using a modified guanidium thiocyanate-phenol-chloroform extraction method. Random hexamer-primed cDNA synthesis was performed in a final volume of 25 µl containing 20 units of RNase inhibitor and 200 units of murine leukemia virus reverse transcriptase (Promega, Madison, WI) at 37°C for 60 min. Then, PCR amplification of human glucose transporter genes was performed with Taq polymerase and oligonucleotide primers. Sequence-specific primers for cytochrome c oxidase-1 (COX-1) and glucose transporters were as follows: COX-1, forward 5'-ACA CGA GCA TAT TTC ACC TCC G-3' and reverse 5'-GGA TTT TGG CGT AGG TTT GGT C-3' (336-bp product); GLUT-1, forward 5'-CCG CAT CAT CTG CCC ACT-3' and reverse 5'-CTG GAT GAC CGA AAA GCT A-3' (352-bp product); GLUT-2, forward 5'-CAA TGA ACC CAA AAC CAA CC-3' and reverse 5'-CAG CTG ATG AAA AGT GCC AA-3' (383-bp product); GLUT-3, forward 5'-ATC ACA GTT GCT ACA ATC GG-3' and reverse 5'-AAT TCC AAC AAC GAT GCC CA-3' (439-bp product); GLUT-4, forward 5'-ACC AAC TGG CCA TTG TTA TC-3' and reverse 5'-CGA AGA TGC TGG TCG AAT AA-3' (415-bp product). Amplification of the human beta -actin gene was performed using commercial primers (Clontech, Palo Alto, CA) to enable semiquantitative normalization. In preliminary experiments, we demonstrated that the product amplification by PCR was linear at the number of cycles. As described, cycle numbers corresponding to the exponential phase of the reaction were determined to be 30 cycles for beta -actin, COX-1, and GLUT-1, 40 cycles for GLUT-2 at 50°C annealing temperature, and 35 cycles at 58°C annealing temperature for GLUT-3 and GLUT-4. Aliquots of PCR reactions were loaded onto a 1.1% agarose gel containing EtBr, electrophoresed, visualized, and quantified by densitometry using Bioprint (Vilber Lourmat, France).

Staining of mitochondria. To study mitochondrial structure and distribution, the control and rho 0 SK-Hep1 cells were incubated with MitoTracker Red (Molecular Probes, Eugene, OR) for 15-45 min at 37°C and then washed three times with prewarmed phosphate-buffered saline (PBS). Cells were fixed for 10 min in 3.7% formaldehyde in PBS at pH 7.4, washed again with PBS, and then incubated with Sytox Green (Molecular Probes) for staining the nucleus. The cell preparations were visualized and photographed in a fluorescence microscope (Carl Zeiss, Jena, Germany).

Measurement of mitochondrial membrane potential. The mitochondrial membrane potential (Delta Psi m) was measured using the fluorescent probe rhodamine 123 (Sigma Chemical, St. Louis, MO) (20). Because rhodamine 123 is a cationic dye, it accumulates in the mitochondria driven by Delta Psi m. Under appropriate loading conditions, the concentration of rhodamine 123 within the mitochondria reaches sufficiently high levels that it quenches its own fluorescence. If the mitochondria depolarize, rhodamine 123 leaks out into the cytoplasm and is associated with a reduction in the amount of quenching. Thus the changes in Delta Psi m are revealed as changes in total fluorescence intensity (8).

The control and rho 0 SK-Hep1 cells were detached from culture dishes with trypsin and washed twice with Krebs-Ringer Henseleit (KRH) buffer (containing in mM: 125 NaCl, 1.2 KH2PO4, 1.2 MgSO4, 6 glucose, 25 HEPES, 2 CaCl2, pH 7.4) and supplemented with 0.2% BSA. Cell suspensions (2 × 105 cells/ml) were incubated in KRH buffer with 10 µg/ml rhodamine 123 at 37°C for 10 min and washed with glucose-free KRH buffer. The measurements of Delta Psi m were carried out in a thermostatically regulated and magnetically stirred fluorimeter cuvette (SFM-25, Bio-Tek Kontron Instruments, Milan, Italy) at a concentration of 2 × 105 cells/ml. The changes in fluorescence intensities were measured at an excitation wavelength of 490 nm and an emission wavelength of 530 nm.

ATP assay. The cellular contents of ATP were measured using the luciferin-luciferase reaction with an ATP bioluminescence assay kit (Sigma). The harvested control and rho 0 SK-Hep1 cells were suspended with KRH buffer containing 0.1 mM glucose and 0.2% BSA. These cell suspensions were incubated in a 37°C shaking water bath. After addition of an assay mixture containing luciferin and luciferase, luminescence was measured immediately in a bioluminometer equipped with an injector (Lumat LB 9501, Berthold, Germany). The amounts of ATP were determined by running an internal standard, expressed as moles per milligram of protein by normalizing to the amount of cellular protein. A Bradford assay was used to determine the protein content.

Glucose uptake using 2-fluoro-2-deoxy-D-[3H]glucose. 2-Fluoro-2-deoxy-D-[5,6-3H]glucose ([3H]FDG; specific activity 30 Ci/mM or 1.1 TBq/mM) was used for the uptake studies (12). Human hepatoma SK-Hep1 cells or the established rho 0 cells (5 × 105 cells/well) were cultured on a 6-well plate in DMEM until 95% confluent. The medium was changed to 1 ml of glucose-free Hanks' balanced salt solution (HBSS), and 1 µCi (37 kBq) of [3H]FDG was added and then incubated at 37°C for 1 h. Addition of ice-cold HBSS stopped the incorporation of [3H]FDG. The cells were washed three times with HBSS and dissolved in 0.5 ml of 0.3 N NaOH with 10% SDS. Aliquots of cell lysates were mixed in 10 ml of scintillation fluid (Hionic Fluor, Packard Instruments, Meriden, CT), and bound 3H activities were measured using a liquid scintillation counter (Packard Instruments). Calibration standards were also used. The tracer uptake was expressed as counts per minute per milligram of cellular protein.

Determination of enzyme activity. COX (EC 1.9.3.1) activity was determined according to the method described by Madden and Storrie (24). Briefly, the enzyme reaction was initiated by the addition of cell lysate to the reaction mixture, and the absorbance change at 550 nm was recorded in a spectrophotometer for a period of 1 min (Spectramax 250, Molecular Devices, Sunnyvale, CA). Succinate dehydrogenase (SDH; EC 1.3.99.1) activity was assayed by determining the absorbance change at 600 nm (28). Lactate dehydrogenase (LDH; EC 1.1.27), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), and hexokinase (EC 2.7.1.1) were assayed as described (10, 17, 26, 33). Specific activities of all enzymes were expressed as milliunits per milligram of protein, where 1 mU was defined as the amount of enzyme that catalyzes the formation of 1 nmol product/min under standard assay conditions.

Western blot analysis. Western blot analysis was performed to estimate expression level of glucose transporter in rho 0 cells. Total cell lysates (50 µg protein) were subjected to 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with goat polyclonal antibodies against human GLUT-1, -3, or -4 (1:1,000; Santa Cruz, Santa Cruz, CA) and with anti-goat IgG conjugated with horseradish peroxidase (1:1,000; Santa Cruz) after three washings in PBS. Bound antibodies were visualized with an enhanced chemiluminescence system (Amersham, Buckinghamshire, England). The antibody against beta -actin was utilized to confirm the equal loading of the sample.

Statistical analysis. Data shown are expressed as means ± SE. Statistical significance was evaluated by paired or unpaired Student's t-test, and P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Establishment of rho 0 SK-Hep1 cells. To establish the mtDNA-depleted cells, the SK-Hep1 human hepatoma cells were treated with the medium containing 0.1 µg/ml EtBr and 50 µg/ml uridine for 10 wk, and the mtDNA contents of the EtBr-treated cells were examined by PCR. Figure 1A shows that mtDNA was not amplified from genomic DNAs of EtBr-treated cells, which are different from the control cells. On the other hand, the beta -actin gene, a nuclear DNA control, was amplified in both control and EtBr-treated cells. This observation demonstrated that the EtBr-treated SK-Hep1 cells were entirely devoid of mtDNA (rho 0 cells). We stained the rho 0 or control SK-Hep1 cells with MitoTracker, a mitochondrial membrane potential-dependent fluorescent dye, to visualize the mitochondrial structure. As shown in Fig. 1B, rho 0 cells lost the reticulum structure of mitochondria in cytoplasm compared with the control SK-Hep1 cells, suggesting that they might lose a functional structure of mitochondria.


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Fig. 1.   Mitochondrial DNA (mtDNA) content and fluorescence microscopic characterization of mitochondria in the human hepatoma SK-Hep1 and mtDNA-depleted (rho 0) SK-Hep1 cells. A: genomic DNAs were isolated from both cells, and then mtDNA and beta -actin nuclear DNA were amplified by PCR. The PCR products were visualized on agarose gel after ethidium bromide (EtBr) staining. B: phase micrographs of the SK-Hep1 (a) and rho 0 SK-Hep1 cells (c) are shown. SK-Hep1 (b) and rho 0 SK-Hep1 (d) cells were stained with MitoTracker Red, a mitochondrial membrane potential-dependent dye, and Sytox Green, a nucleic acid-staining dye. Original magnification is ×1,000.

Loss of Delta Psi m in rho 0 cells. To confirm whether rho 0 cells lost oxidative phosphorylation activity, we measured Delta Psi m using rhodamine 123. In control SK-Hep1 cells, an application of 10 mM glucose hyperpolarized Delta Psi m, and the subsequent treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 µM), a mitochondrial uncoupler, rapidly depolarized Delta Psi m (n = 10). However, the rho 0 cells did not hyperpolarize Delta Psi m upon glucose application, indicating that the rho 0 cells may be unable to utilize glucose metabolites as substrates for ATP production through the tricarboxylic acid cycle. Furthermore, the rho 0 cells failed to change Delta Psi m when treated with FCCP alone, which reflected the amplitude of resting Delta Psi m (n = 6; Fig. 2A). Figure 2B shows the quantitative changes of Delta Psi m in the control or rho 0 cells by either glucose or FCCP. This result suggests that the rho 0 cells lack the basal proton gradient across the mitochondrial membrane as well as the glucose-stimulated gradient generation.


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Fig. 2.   Changes of mitochondrial membrane potential (Delta Psi m). Cell suspensions were loaded with rhodamine 123 (10 µg/ml for 10 min) in Krebs-Ringer Henseleit (KRH) buffer. A: representative traces illustrating that Delta Psi m changes were evoked by the addition (arrows) of glucose (10 mM) and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 µM) in SK-Hep1 (n = 10) and its rho 0 cells (n = 6). B: percent changes of fluorescence by glucose and FCCP are expressed as means ± SE; **P < 0.01.

Decreased COX activity in rho 0 cells. The three subunits of COX complex (COX-1, -2, and -3) in the mitochondrial electron transfer system are encoded by mtDNA, whereas the other 10 subunits are encoded by nuclear DNA. We determined the steady-state mRNA levels of COX-1 in the control and rho 0 cells by RT-PCR. As expected, the COX-1 mRNA was nearly absent, whereas beta -actin was normally expressed in rho 0 cells (Fig. 3). The total cellular enzyme activity of COX was found to be significantly decreased in rho 0 cells compared with that in the control cells (2.4 ± 0.6 vs. 1.0 ± 0.7 mU/mg protein; n = 4, P < 0.01). In contrast, the specific activity of SDH, of which all subunits are nuclear DNA encoded, were slightly increased by mtDNA depletion (353 ± 15 vs. 400 ± 28 mU/mg protein; n = 5, P < 0.05).


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Fig. 3.   Steady-state mRNA levels of cytochrome c oxidase (COX)-1. Total RNAs were isolated from SK-Hep1 and rho 0 SK-Hep1 cells, and the mRNA levels of COX-1 and beta -actin were determined by RT-PCR, as described in MATERIALS AND METHODS. The products were visualized on agarose gel after EtBr staining.

Attenuated ATP production in rho 0 cells. There are several reports showing that the adipocytes and muscle cells initially decrease the intracellular ATP content by inhibiting oxidative phosphorylation but subsequently recovering it (4). Results from mtDNA depletion were different from this. In the case of rho 0 SK-Hep1 cells, the resting intracellular ATP contents were decreased 80% compared with the control cells [0.35 ± 0.03 (n = 13) vs. 1.84 ± 0.09 nmol/mg protein (n = 11), P < 0.01]. Furthermore, the glucose-stimulated ATP production was also largely attenuated in rho 0 cells. The application of glucose (10 mM) for 10 min produced 0.44 ± 0.09 nmol ATP/mg protein in the control SK-Hep1 cells, whereas only 0.04 ± 0.07 nmol ATP/mg protein was produced in rho 0 cells (Fig. 4).


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Fig. 4.   Intracellular ATP contents at basal state and after glucose stimulation (10 mM; arrows) were compared between SK-Hep1 (, n = 13) and its rho 0 cells (open circle , n = 11), which were measured by bioluminometer using luciferin-luciferase reaction. ATP contents were normalized to cellular protein and expressed as nanomoles per milligram of cellular protein. Data are expressed as means ± SE.

Reduced glucose uptake and glucose transporter expression in rho 0 cells. To investigate whether mtDNA depletion impairs glucose utilization, glucose uptake was assessed using [3H]FDG, a radiolabeled, nondegradable glucose (12). The resting and insulin-stimulated [3H]FDG uptakes in rho 0 cells were decreased by ~30% compared with the control cells (Fig. 5).


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Fig. 5.   Basal and insulin-stimulated glucose uptake. Both SK-Hep1 and its rho 0 cells (n = 11) were incubated with 2-fluoro-2-deoxy-D-[3H]glucose ([3H]FDG), and then cellular 3H activities were determined by a liquid scintillation counter. Data are expressed as means ± SE; **P < 0.01.

It is possible that the decrease in glucose uptake may result from an alteration of glucose transporter expressions. The steady-state levels of mRNA and proteins of the glucose transporter isoforms, GLUT-1, -2, -3, and -4, were determined by RT-PCR and Western blot. The levels of GLUT-1, GLUT-3, and GLUT-4 mRNA in rho 0 cells were reduced by 10.2 ± 4.2, 35.5 ± 8.9, and 57.2 ± 8.4%, respectively, compared with those in control cells (Fig. 6). However, the mRNA of GLUT-2 was not present in either control or rho 0 SK-Hep1 cells (data not shown). To a similar degree, the protein levels of GLUT-1, GLUT-3, and GLUT-4 were attenuated in rho 0 cells (Fig. 7). These data demonstrate that the mtDNA depletion downregulated the expression of glucose transporters, which were nuclear encoded, resulting in a reduction of glucose utilization.


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Fig. 6.   Steady-state mRNA levels of glucose transporters. Total RNA was isolated from SK-Hep1 or its rho 0 cells. The steady-state mRNA levels of GLUT-1 (n = 9), GLUT-3 (n = 5), GLUT-4 (n = 4), and beta -actin were determined by RT-PCR. A: representative pictures are shown. GLUT-2 was not expressed in either group of cells. B: band intensities of the transporters were normalized to the intensity of beta -actin and expressed as the ratio (GLUT/beta -actin). Data are means ± SE; *P < 0.05, **P < 0.01.



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Fig. 7.   Protein expression levels of glucose transporters. Total cell lysates (50 µg protein) were subjected to 10% SDS-PAGE. Western blot analysis was performed using goat polyclonal antibody against human glucose transporters (GLUT-1, -3, and -4). Bound antibodies were visualized with enhanced chemiluminescence. The antibody against human beta -actin was utilized to confirm the equal loading of the samples.

Activities of glucose-metabolizing enzymes. The effects of mtDNA depletion on glycolysis and pentose phosphate shunt were determined by cellular activities of GAPDH and G6PDH. The specific activities of GAPDH and G6PDH in rho 0 cells were reduced to 75 and 30% of the control cells, respectively (Table 1). Interestingly, the activity of hexokinase in rho 0 cells was only 10% of the control cells. The activity of LDH, an indicator for the intracellular NADH accumulation due to dysfunction of the respiratory chain, was 53% enhanced in rho 0 cells. The increased LDH activity in rho 0 cells implies that the minimal energy production for cell survival might be achieved via anaerobic metabolism.

                              
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Table 1.   Specific activities of enzymes involved in glucose metabolism


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondria are intracellular organelles that are the site of oxidative phosphorylation and the primary source of cellular energy (34). It is known that mutations, deletions, and insertions of mtDNA result in mitochondrial dysfunction associated with the pathogenesis of various mitochondrial diseases, including DM (3). There are also several lines of evidence to support the idea that the alterations of mtDNA quantity may cause mitochondrial diseases. First, depletions of mtDNA in muscle, liver, and kidney were reported in fatal mitochondrial encephalopathies (27). Second, streptozotocin, one of the diabetogenic agents, reduced the levels of mtDNA content and its transcripts in pancreatic islets (11). Third, beta -cells of adult Goto-Kakizaki rats, a genetic model of defective insulin secretion and hyperglycemia, had a significantly smaller mitochondrial volume compared with the beta -cells of control rats (29). Fourth, mtDNA depletion inhibited glucose-stimulated increases of the intracellular free Ca2+ concentration and insulin secretion in mouse insulinoma cells, which led to glucose intolerance and hyperglycemia (31). Finally, in our laboratory, the decrease of mtDNA content preceded the development of diabetes (22), suggesting that the quantitative reduction of mtDNA be considered as one of the possible causative factors, not a consequence, of diabetes.

In this study, we established the mtDNA-depleted rho 0 cells using SK-Hep1, a rapidly proliferating human hepatoma cell, to study whether decreased mtDNA might disturb the glucose metabolism in liver cells, resulting in diabetes. First, the rho 0 SK-Hep1 cells showed a slower growth rate than the parental SK-Hep1 cells, which was similar to the case of rho 0 HeLa cells (7). Because of the low levels of intracellular ATP content and G6PDH, which are determinants of NADPH production and are essential for cell growth, it may be natural for rho 0 cells to grow slowly (32). However, growth retardation was not observed in all mtDNA-depleted cells; the growth rate of human rho 0 osteosarcoma cells was normal (21). This finding indicates that different cells exhibit different sensitivities to intracellular changes elicited by mtDNA depletion.

Next, we determined which mitochondrial functions were altered by mtDNA depletion. It was interesting that the rho 0 cell showed no response to FCCP in Delta Psi m, indicating that the rho 0 cells lose the proton gradient across the mitochondrial membrane. There are reports of discrepancies on Delta Psi m of rho 0 cells, although the impaired oxidative phosphorylation may account for the loss of Delta Psi m. Kennedy et al. (20) reported that Delta Psi m was abolished in rho 0 insulinoma cells, whereas Buchet and Godinot (7) reported that Delta Psi m was maintained in rho 0 HeLa cells through the functional F1-ATPase and adenine nucleotide translocator. It is unknown whether rho 0 insulinoma or SK-Hep1 cells contain normal activities of F1-ATPase and adenine nucleotide translocator.

Defects in glucose uptake into muscles and adipose tissues are generally accepted as one of the major characteristics of type 2 DM. A decrease in hepatic glucose uptake was also reported in type 2 diabetic patients and animal models (5, 30). The basal [3H]FDG uptake into rho 0 cells was decreased by 30% compared with the control cells, whereas insulin-stimulated uptake did not occur in either rho 0 or control cells. Because GLUT-4 is known to play a major role in insulin-responsive glucose transport in peripheral tissues, including muscles and adipose tissues, GLUT-4 might contribute to total glucose uptake relatively less than others (19) in SK-Hep1 cells. This is consistent with the fact that GLUT-3 and GLUT-4 are expressed at a relatively lower level in SK-Hep1 cells than GLUT-1. GLUT-1, which is dominant in fetal hepatocytes (15), is the most abundant type of glucose transporter in SK-Hep1 cells. We found that the mRNA of GLUT-2, a major glucose transporter in normal adult hepatocytes, was not detected in control SK-Hep1 cells, as it has been reported that the expression of GLUT-2 was severely decreased throughout hepatocarcinogenesis (15). Also, depletion of mtDNA significantly attenuated the expression of all types of glucose transporters that are encoded by nuclear DNA. Further studies are needed to show how alterations of mtDNA could affect the expression of nuclear DNA-encoded proteins involved in glucose transport but not a structural protein like beta -actin.

It is possible that the decreased glucose uptake reduces the glucose supply for enzymes involved in glucose metabolic pathways. These enzymes may be inactivated or downregulated. As expected, we found that, in rho 0 cells, the activities of hexokinase, GAPDH, and G6PDH were decreased by 90, 25, and 70%, respectively. These data suggest that the depletion of mtDNA decreased glucose utilization by suppressing glucose metabolism in addition to reducing glucose uptake. Furthermore, the attenuated activities of glycolytic enzymes could consequently reduce ATP production, which may aggravate ATP depletion in rho 0 cells in a vicious cycle.

It is interesting to note the large decrease of hexokinase activity in rho 0 cells. In contrast to other glycolytic enzymes, hexokinase is known to be associated with the mitochondrial outer membrane through its interaction with porin and uses intramitochondrial ATP supplied by oxidative phosphorylation as a substrate for glucose phosphorylation (9). Moreover, the activation of hexokinase depends on a contact site-specific structure of the pore, which is voltage dependent and influenced by the electrical potential of the mitochondrial inner membrane (13). It has been reported that mitochondria lacking a membrane potential induced by a mitochondrial uncoupler such as dinitrophenol decreases the contacts and hexokinase activity in hepatocytes (6). We also observed that the hexokinase protein was significantly decreased in mitochondrial fraction from rho 0 cells, and this was determined by Western blotting assay (unpublished data). Therefore, we can infer that the defects in intramitochondrial ATP production and depolarized Delta Psi m in rho 0 cells could inactivate hexokinase. Furthermore, the decreased hexokinase activity in rho 0 cells may be one of the fundamental causes of the disturbed glucose metabolism.

This study demonstrates for the first time that mtDNA depletion may disturb the cellular capacity for glucose utilization, at least in liver cells, although the study was performed in a single cell line and not over a wide variety of cell types. Further studies on other changes in various types of mtDNA-depleted cell lines would be helpful in extending our knowledge about the pathogenic role of quantitative changes in mtDNA.


    ACKNOWLEDGEMENTS

The authors thank Dr. Soo-Young Park for precious support on this work.


    FOOTNOTES

This study was supported by Grant no. 00-4 of the National Institute of Health, Seoul, Korea.

Address for reprint requests and other correspondence: Y. K. Pak, Div. of Metabolic Disease, Dept. of Biomedical Sciences, National Institute of Health, 5 Nokbun-Dong, Eunpyung-Ku, Seoul, Korea 122-701 (E-mail: ymkimpak{at}nih.go.kr).

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.

Received 28 June 2000; accepted in final form 5 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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