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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 0 SK-Hep1 human hepatoma cells, whose mtDNA was
depleted by long-term exposure to ethidium bromide. The
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
0 cells compared with parental
+ 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 (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
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
0
human hepatoma cell line, which lost the ability of oxidative phosphorylation. We observed that cellular ATP production and glucose
uptake were attenuated in
0 cells. Glucose transporter
expression and glucose metabolizing enzyme activities in
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (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 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.
Staining of mitochondria.
To study mitochondrial structure and distribution, the control and
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 (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
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
m are revealed as changes in total fluorescence
intensity (8).
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 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 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 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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Establishment of 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
-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 (
0 cells). We
stained the
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,
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.
|
Loss of m in
0 cells.
To confirm whether
0 cells lost oxidative
phosphorylation activity, we measured
m using
rhodamine 123. In control SK-Hep1 cells, an application of 10 mM
glucose hyperpolarized
m, and the subsequent
treatment with carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 µM), a
mitochondrial uncoupler, rapidly depolarized
m (n = 10). However, the
0 cells did not
hyperpolarize
m upon glucose application, indicating that the
0 cells may be unable to utilize glucose
metabolites as substrates for ATP production through the tricarboxylic
acid cycle. Furthermore, the
0 cells failed to change
m when treated with FCCP alone, which reflected the
amplitude of resting
m (n = 6; Fig.
2A). Figure 2B
shows the quantitative changes of
m in the control or
0 cells by either glucose or FCCP. This result suggests
that the
0 cells lack the basal proton gradient across
the mitochondrial membrane as well as the glucose-stimulated gradient
generation.
|
Decreased COX activity in 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
0
cells by RT-PCR. As expected, the COX-1 mRNA was nearly absent, whereas
-actin was normally expressed in
0 cells (Fig.
3). The total cellular enzyme activity of
COX was found to be significantly decreased in
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).
|
Attenuated ATP production in 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
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
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
0 cells (Fig. 4).
|
Reduced glucose uptake and glucose transporter expression in
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
0 cells were decreased by ~30% compared with
the control cells (Fig. 5).
|
|
|
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 0 cells were
reduced to 75 and 30% of the control cells, respectively (Table 1). Interestingly, the activity of
hexokinase in
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
0 cells. The increased LDH activity in
0 cells implies that the minimal energy production for
cell survival might be achieved via anaerobic metabolism.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, -cells of adult Goto-Kakizaki rats, a
genetic model of defective insulin secretion and hyperglycemia, had a
significantly smaller mitochondrial volume compared with the
-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 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
0 SK-Hep1 cells
showed a slower growth rate than the parental SK-Hep1 cells, which was
similar to the case of
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
0 cells to grow slowly
(32). However, growth retardation was not observed in all
mtDNA-depleted cells; the growth rate of human
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 0 cell showed no
response to FCCP in
m, indicating that the
0 cells lose the proton gradient across the
mitochondrial membrane. There are reports of discrepancies on
m of
0 cells, although the impaired
oxidative phosphorylation may account for the loss of
m. Kennedy et al. (20) reported that
m was abolished in
0 insulinoma cells,
whereas Buchet and Godinot (7) reported that
m was maintained in
0 HeLa cells
through the functional F1-ATPase and adenine nucleotide translocator.
It is unknown whether
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 0 cells was decreased
by 30% compared with the control cells, whereas insulin-stimulated
uptake did not occur in either
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
-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 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
0 cells in a vicious cycle.
It is interesting to note the large decrease of hexokinase activity in
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
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
m in
0
cells could inactivate hexokinase. Furthermore, the decreased hexokinase activity in
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Antonetti, DA,
Reynet C,
and
Kahn CR.
Increased expression of mitochondrial-encoded genes in skeletal muscle of humans with diabetes mellitus.
J Clin Invest
95:
1383-1388,
1995[ISI][Medline].
2.
Ballinger, SW,
Shoffner JM,
Gebhart S,
Koontz DA,
and
Wallace DC.
Mitochondrial diabetes revisited.
Nat Genet
7:
458-459,
1994[ISI][Medline].
3.
Ballinger, SW,
Shoffner JM,
Hedaya EV,
Trounce I,
Polak MA,
Koontz DA,
and
Wallace DC.
Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion.
Nat Genet
1:
11-15,
1992[ISI][Medline].
4.
Bashan, N,
Burdett E,
Guma A,
Sargeant R,
Tumiati L,
Liu Z,
and
Klip A.
Mechanism of adaptation of glucose transporters to changes in the oxidative chain of muscle and fat cells.
Am J Physiol Cell Physiol
264:
C430-C440,
1993
5.
Basu, A,
Basu R,
Shah P,
Vella A,
Johnson CM,
Nair KS,
Jensen MD,
Schwenk WF,
and
Rizza RA.
Effects of type 2 diabetes on the ability of insulin and glucose to regulate splanchnic and muscle glucose metabolism: evidence for a defect in hepatic glucokinase activity.
Diabetes
49:
272-283,
2000[Abstract].
6.
Brdiczka, D.
Function of the outer mitochondrial compartment in regulation of energy metabolism.
Biochim Biophys Acta
1187:
264-269,
1994[ISI][Medline].
7.
Buchet, K,
and
Godinot C.
Functional F1-ATPase essential in maintaining growth and membrane potential of human mitochondrial DNA-depleted rho degrees cells.
J Biol Chem
273:
22983-22989,
1998
8.
Buckler, KJ,
and
Vaughan-Jones RD.
Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells.
J Physiol (Lond)
513:
819-833,
1998
9.
Cesar, MD,
and
Wilson JE.
Further studies on the coupling of mitochondrially bound hexokinase to intramitochondrially compartmented ATP, generated by oxidative phosphorylation.
Arch Biochem Biophys
350:
109-117,
1998[ISI][Medline].
10.
DeMoss, RD.
Glucose-6-phosphate and 6-phosphogluconic dehydrogenase from Leuconostoc mesenteroides. In:
In: Methods in Enzymology, edited by Colowick SP,
and Kaplan NO.. New York: Academic, 1955, vol. 1, p. 328.
11.
Eizirik, DL,
Sandler S,
Ahnstrom G,
and
Welsh M.
Exposure of pancreatic islets to different alkylating agents decreases mitochondrial DNA content but only streptozotocin induces long-lasting functional impairment of -cells.
Biochem Pharmacol
42:
2275-2282,
1991[ISI][Medline].
12.
Gallagher, BM,
Fowler JS,
Gutterson NI,
MacGregor RR,
Wan CN,
and
Wolf AP.
Metabolic trapping as a principle of oradiopharmaceutical design: some factors responsible for the biodistribution of [18F]2-deoxy-2-fluoro-D-glucose.
J Nucl Med
19:
1154-1161,
1978[ISI][Medline].
13.
Gerbitz, KD,
Gempel K,
and
Brdiczka D.
Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit.
Diabetes
45:
113-126,
1996[Abstract].
14.
Gerbitz, KD,
van den Ouweland JM,
Maassen JA,
and
Jaksch M.
Mitochondrial diabetes mellitus: a review.
Biochim Biophys Acta
1271:
253-260,
1995[ISI][Medline].
15.
Grobholz, R,
Hacker HJ,
Thorens B,
and
Bannasch P.
Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.
Cancer Res
53:
4204-4211,
1993[Abstract].
16.
Hayakawa, T,
Noda M,
Yasuda K,
Yorifuji H,
Taniguchi S,
Miwa I,
Sakura H,
Terauchi Y,
Hayashi J,
Sharp GW,
Kanazawa Y,
Akanuma Y,
Yazaki Y,
and
Kadowaki T.
Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line HC9.
J Biol Chem
273:
20300-20307,
1998
17.
Hirota, Y,
Cohen EM,
and
Bing OH.
Lactate dehydrogenase and isoenzyme changes in rats with experimental thiamine deficiency.
Metabolism
25:
1-8,
1976[ISI][Medline].
18.
Kadowaki, T,
Kadowaki H,
Mori Y,
Tobe K,
Sakuta R,
Suzuki Y,
Tanabe Y,
Sakura H,
Awata T,
and
Goto Y.
A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA.
N Engl J Med
330:
962-968,
1994
19.
Kahn, BB.
Lilly lecture 1995. Glucose transport: pivotal step in insulin action.
Diabetes
45:
1644-1654,
1996[Abstract].
20.
Kennedy, ED,
Maechler P,
and
Wollheim CB.
Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells.
Diabetes
47:
374-380,
1998[Abstract].
21.
King, MP,
and
Attardi G.
Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation.
Science
246:
500-503,
1989[ISI][Medline].
22.
Lee, HK,
Song JH,
Shin CS,
Park DJ,
Park KS,
Lee KU,
and
Koh CS.
Decreased mitochondrial DNA content in peripheral blood precedes the development of non-insulin-dependent diabetes mellitus.
Diabetes Res Clin Pract
42:
161-167,
1998[ISI][Medline].
23.
Li, SR,
Baroni MG,
Oelbaum RS,
Stock J,
and
Galton DJ.
Association of genetic variant of the glucose transporter with non-insulin-dependent diabetes mellitus.
Lancet
2:
368-370,
1988[ISI][Medline].
24.
Madden, EA,
and
Storrie B.
The preparative isolation of mitochondria from Chinese hamster ovary cells.
Anal Biochem
163:
350-357,
1987[ISI][Medline].
25.
Martin, BC,
Warram JH,
Krolewski AS,
Bergman RN,
Soeldner JS,
and
Kahn CR.
Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study.
Lancet
340:
925-929,
1992[ISI][Medline].
26.
Miwa, I,
Mita Y,
Murata T,
Okuda J,
Sugiura M,
Hamada Y,
and
Chiba T.
Utility of 3-O-methyl-N-acetyl-D-glucosamine, an N-acetylglucosamine kinase inhibitor, for accurate assay of glucokinase in pancreatic islets and liver.
Enzyme Protein
48:
135-142,
1994[ISI][Medline].
27.
Moraes, CT,
Shanske S,
Tritschler HJ,
Aprille JR,
Andreetta F,
Bonilla E,
Schon EA,
and
DiMauro S.
mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases.
Am J Hum Genet
48:
492-501,
1991[ISI][Medline].
28.
Owen, P,
and
Freer JH.
Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus.
Biochem J
120:
237-243,
1970[ISI][Medline].
29.
Serradas, P,
Giroix MH,
Saulnier C,
Gangnerau MN,
Borg LA,
Welsh M,
Portha B,
and
Welsh N.
Mitochondrial deoxyribonucleic acid content is specifically decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of non-insulin-dependent diabetes.
Endocrinology
136:
5623-5631,
1995[Abstract].
30.
Shiba, Y,
Yamasaki Y,
Kubota M,
Matsuhisa M,
Tomita T,
Nakahara I,
Morishima T,
Kawamori R,
and
Hori M.
Increased hepatic glucose production and decreased hepatic glucose uptake at the prediabetic phase in the Otsuka Long-Evans Tokushima fatty rat model.
Metabolism
47:
908-914,
1998[ISI][Medline].
31.
Soejima, A,
Inoue K,
Takai D,
Kaneko M,
Ishihara H,
Oka Y,
and
Hayashi JI.
Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6.
J Biol Chem
271:
26194-26199,
1996
32.
Tian, WN,
Braunstein LD,
Pang J,
Stuhlmeier KM,
Xi QC,
Tian X,
and
Stanton RC.
Importance of glucose-6-phosphate dehydrogenase activity for cell growth.
J Biol Chem
273:
10609-10617,
1998
33.
Velick, SF.
Glyceraldehyde-3-phosphate dehydrogenase.
In: The Enzymes, edited by Boyer PD,
Lardy H,
and Myrback K.. New York: Academic, 1961, vol. 7, p. 243.
34.
Wallace, DC.
Mitochondrial genetics: a paradigm for aging and degenerative diseases?
Science
256:
628-632,
1992[ISI][Medline].
35.
Zylber, E,
Vesco C,
and
Penman S.
Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide.
J Mol Biol
44:
195-204,
1969[ISI][Medline].