In Vivo Role of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Regulation of Intracellular Redox State and
Accumulation of Abdominal Adipose Tissue*
Amos
Gaikwad
,
Delwin J.
Long II
,
Janet L.
Stringer, and
Anil
K.
Jaiswal§
From the Department of Pharmacology, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, February 4, 2001, and in revised form, March 28, 2001
 |
ABSTRACT |
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a
flavoprotein that utilizes NAD(P)H as an electron donor, catalyzing the
two-electron reduction and detoxification of quinones and their
derivatives. NQO1
/
mice deficient in NQO1 activity and protein were
generated in our laboratory (Rajendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J. P., Gonzalez, F. J.,
and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389). Mice lacking a functional NQO1 gene (NQO1
/
) were born
normal and reproduced adeptly as the wild-type NQO1+/+ mice. In the
present report, we show that NQO1
/
mice exhibit significantly lower
levels of abdominal adipose tissue as compared with the wild-type mice.
The NQO1
/
mice showed lower blood levels of glucose, no change in
insulin, and higher levels of triglycerides,
-hydroxy butyrate,
pyruvate, lactate, and glucagon as compared with wild-type mice.
Insulin tolerance test demonstrated that the NQO1
/
mice are insulin
resistant. The NQO1
/
mice livers also showed significantly higher
levels of triglycerides, lactate, pyruvate, and glucose. The liver
glycogen reserve was found decreased in NQO1
/
mice as compared with
wild-type mice. The livers and kidneys from NQO1
/
mice also showed
significantly lower levels of pyridine nucleotides but an increase in
the reduced/oxidized NAD(P)H:NAD(P) ratio. These results suggested that
loss of NQO1 activity alters the intracellular redox status by
increasing the concentration of NAD(P)H. This leads to a reduction in
pyridine nucleotide synthesis and reduced glucose and fatty acid
metabolism. The alterations in metabolism due to redox changes result
in a significant reduction in the amount of abdominal adipose tissue.
 |
INTRODUCTION |
NAD(P)H:quinone oxidoreductase 1 (NQO1)1 is a 274-amino acid
flavoprotein that catalyzes the two-electron reduction and
detoxification of quinones and their derivatives (1-3). The cytosolic
NQO1 activities, purified from rat liver and human adipose tissue, have
been characterized and cloned (1-3). NQO1 utilizes both NADH and NADPH
as electron donors (1-3). The two-electron reduction of quinones does
not result in the formation of free radicals (semiquinones) and highly reactive oxygen species, hence protecting cells against the adverse effects of quinones and their derivatives (1-3). As a protective agent, NQO1 activity has been shown to prevent the formation of highly
reactive quinone metabolites (4), detoxify benzo(a)pyrene quinone (5), and reduce chromium (VI) toxicity (6). Recently, NQO1 was
also shown to reduce benzo(a)pyrene and
benzo(a)pyrene quinone induced mutagenicity (7, 8).
NQO1 activity is present in all tissues but at different levels (1-3).
Various investigators have observed large variations in NQO1 activity
between individuals, in different tissues from the same individual, and
between normal/tumor tissues (1-3). It is generally accepted that
tumor tissues and cells of hepatic and colonic origin express higher
levels of NQO1, as compared with normal tissues and cells of similar
origins (1-3). The normal tissue that surrounds the hepatic tumors
also expresses higher levels of the NQO1 gene, presumably to play an
unknown role in tumor progression (9). NQO1 gene expression is induced
in response to xenobiotics, antioxidants, oxidants, heavy metals, UV
light, and ionizing radiation (1-3, 10). Interestingly, NQO1 is part of an electrophilic and/or oxidative stress-induced cellular defense mechanism that includes the induction of more than two dozen genes (1-3). The coordinated induction of these genes, including NQO1, presumably provides cellular protection against free radical damage, oxidative stress, and neoplasia.
Despite the large volume of knowledge about NQO1, the in
vivo role of NQO1 remains unknown. To examine the in
vivo role of NQO1, we used targeted gene disruption to generate
NQO1
/
mice (11). These mice are born normal and reproduce normally.
However, the NQO1
/
mice exhibited increased toxicity to menadione
when compared with the wild-type mice. In the present study, we
demonstrate that NQO1
/
mice exhibit a significant reduction or
absence of abdominal adipose tissue, as compared with the wild-type
mice. We also demonstrate that several metabolic pathways are altered in NQO1
/
mice as compared with wild-type mice. This was evident from lower blood levels of glucose and higher levels of triglycerides,
-hydroxybutyrate, lactate, pyruvate, and glucagon in
NQO1
/
mice as compared with wild-type mice. The NQO1
/
mice
livers showed lower levels of glycogen but higher levels of glucose, triglycerides, lactate, and pyruvate. We further demonstrate that NQO1
/
mice are insulin resistant as compared with wild-type mice.
In addition, the NQO1
/
mice livers and kidneys showed decreased
pyridine nucleotides but higher NAD(P)H:NAD(P) ratio as compared with
wild-type mice. These results suggest that NQO1 plays a significant
role in regulation of intracellular redox status and hence metabolic
pathways leading to the accumulation of abdominal adipose tissue.
 |
EXPERIMENTAL PROCEDURES |
Breeding of Wild-type and NQO1
/
Mice--
C57BL6 NQO1
/
and wild-type mice were bred in our laboratory (11). The animals were
housed in polycarbonate cages, maintained with a 12-h light/dark cycle,
a temperature of 24 ± 2 °C, a relative humidity of 55 ± 10%, and a negative atmospheric pressure. The mice were fed standard
rodent chow and acidified tap water ad libitum.
Twenty-four-, 48-, and 72-week-old male and female mice were used in
the present studies. However, because of the similarity in the results
the data are shown only for male mice. Animals received humane care
throughout the experiment according to AALAC guidelines for animal welfare.
Collection of Blood and Tissues for Analysis--
Mice were
anesthetized by a brief exposure to isoflurane (VEDCO Inc., St.
Joseph, MO). The mice were then given a 0.25-ml intraperitoneal
injection of 50% urethane. Blood was extracted from the hearts with a
1-ml EDTA-coated syringe/22G1 needle and placed in Eppendorf tubes. The
tubes were inverted several times to ensure uniform clotting. After a
20-min incubation at room temperature, the tubes were centrifuged at
14,000 rpm for 20 min. The serum was withdrawn from the tubes and
frozen at
20 °C until analysis. Blood and tissue samples were
taken at the same time of the day. In related experiments, animals were
fasted for 16 h and blood was drawn for analysis as described earlier.
Serum Analysis--
The glucose and triglyceride levels were
analyzed in the serum samples using a Cobas Mira System and Sigma
reagents.
-Hydroxybutyrate, lactate, and pyruvate levels were
measured using Sigma Diagnostic Kits and protocol supplied by the
manufacturer. Linco Research Inc. (St. Charles, MO) using a
sensitive radioimmunoassay kit for the determination of concentration
of glucagon and insulin in the serum.
Insulin Tolerance Test--
The insulin tolerance test was
performed by previously described method (12). Seventy-two-week-old
wild-type and age-matched NQO1
/
mice were used. The mice were
fasted for 16 h but given water ad libitum. Insulin
(0.15 IU/kg body weight) was administered by intraperitonial injection
of insulin to the fasted mice. The blood was drawn by retro-orbital
bleeding and analyzed for blood glucose using the Accu Chek kit (Roche
Molecular Biochemicals).
NQO1 Activity and Western Blot Analysis--
The various tissues
(liver and kidney) from 24-week-old wild-type and NQO1
/
mice were
homogenized in 50 mM Tris (pH 7.4) containing 0.25 M sucrose. This homogenate was centrifuged at 105,000 × g for 1 h to obtain cytosolic fractions.
Dicoumarol-sensitive NQO1 activity was determined in all cytosolic
fractions by a previously described method (13). The various cytosolic
fractions were also analyzed for NQO1 protein by Western blotting using
antibodies against the purified rat liver NQO1 protein (13). The rat
NQO1 antibody is known to cross-react with mouse and human proteins (13-14). Western blots were developed with ECL (Amersham Pharmacia Biotech) reagents by the procedures suggested by the manufacturer.
Collection and Processing of Mice Tissues--
Two different
procedures were used to collect and process the tissues for
determination of various metabolites and oxidized and reduced pyridine
nucleotide levels because of sensitivity of these molecules. In the
classical method, the mice were sacrificed by cervical dislocation
(15). The tissues were surgically removed and instantly
frozen in liquid nitrogen and weighed in frozen condition (15). In the
freeze-clamping method, the mice were anesthetized with sodium
pentobarbital, tissues perfused, and prepared using freeze-clamping
device (16, 17).
Quantitation of Various Metabolites in Liver--
The frozen
liver was weighed, cut into pieces, and homogenized in ice-cold buffer
containing 250 mM sucrose, 50 mM Tris-Cl (pH
7.6) and a set of protease inhibitors containing 0.1 mM
phenylmethanesulfonyl fluoride, 0.5 mM EDTA, 1 mg/ml
benzamidine-Cl, and 10 µM leupeptin. The homogenate was
extracted with 5% trichloroacetic acid (final concentration). The
sample was allowed to precipitate for 30 min on ice and a clear
supernatant was obtained by centrifuging the samples at 12,000 rpm for
20 min at 4 °C. The supernatant was neutralized to pH 7.6 using
Tris-Cl buffer. Lactate, pyruvate, and
-hydroxybutyrate were
measured in the various samples using the Sigma Diagnostic Kits and the
manufacturer's protocols. Glucose and triglycerides were estimated
using a Cobas Mira System and Sigma Diagnostic Kit. Glycogen reserve in
the liver tissues were measured by a procedure as previously described
(18-19). The classical and freeze-clamping techniques yielded more or
less similar results for various metabolites. Therefore, the values
from both these methods were combined.
Measurement of Pyridine Nucleotides in Tissues--
The mice
liver and kidney tissues collected by classical and freeze-clamping
methods were homogenized in chilled buffer containing 200 mM KCN, 1 mM bathophenanthroline, and 60 mM KOH using a Potter-Elvejham homogenizer system. The
pyridines were extracted and analyzed from these tissues by procedures
as previously described (15). Briefly, the homogenate was extracted
rapidly with chloroform several times until minimal precipitate was
observed at the interface of the buffer and chloroform. The supernatant
was then passed through a 0.45-µm Ultrafree-MC filtration device
(Millipore) by centrifuging at 5000 rpm for 10 min at 4 °C. This
removes any residual DNA/protein and also serves as a filter before the
samples are loaded onto the HPLC column. The various pyridine
nucleotides (NAD, NADH, NADP, and NADPH) were separated, analyzed, and
quantitated using a C18 chromatography column (Waters) and HPLC
(Waters). The mobile phase consisted of 50 mM potassium
phosphate buffer (pH 7.05):acetonitrile (97:3) as suggested by the
manufacturer. The RF values of the NAD, NADP, NADH,
and NADPH standards were calculated and employed for quantitation of
the pyridine nucleotides in the various samples under identical
conditions. The classical and freeze-clamping techniques yielded quite
similar results. Therefore, the values from both these methods were combined.
 |
RESULTS |
Wild-type (NQO1+/+) and NQO1
/
mice were analyzed for NQO1
activity and protein levels (Fig. 1). The
highest amount of NQO1 activity was observed in the kidney, followed by
the liver and abdominal adipose tissue. The NQO1 activities were
similar for male and female mice, therefore, the data has been
combined. NQO1 activity was not detected in the kidney or abdominal
adipose tissue from NQO1
mice. However, the livers from NQO1
/
mice contained a small amount (<15% of wild type) of NQO1 activity.
Western blot analysis did not detect any NQO1 protein in these three
tissues (liver, kidney, and abdominal adipose), but did detect NQO1
protein in similar tissues from wild-type (NQO1+/+) mice. The small
amounts of NQO1 activity detected in the liver of NQO1
/
mice may be due to an isoenzymic form of NQO1, since the NQO1 protein was not
detected in Western analysis of NQO1
/
liver tissue (Fig. 1). A
comparison of total body weight of wild-type and NQO1
/
mice was
performed. The results indicated that NQO1
/
mice were significantly
lighter in weight as compared with wild-type mice at all the three (24, 48, and 72 weeks old) ages of mice studied (Table
I).

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Fig. 1.
NQO1 activity and Western blot analysis of
wild-type and NQO1 / mice. The 24-week-old wild-type and
NQO1 / male mice were used. The liver, kidney, and adipose tissues
were collected and homogenized in appropriate buffer. The homogenates
were centrifuged at 100,000 × g for 1 h and
cytosolic fractions collected. The cytosolic fractions were analyzed
for NQO1 activity and NQO1 protein by Western analysis. The Western
blot was probed with polyclonal NQO1 antibodies against rat NQO1
protein.
|
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The results of the analysis of the abdominal adipose tissue in 24-, 48-, and 72-week-old wild-type and NQO1
/
mice are shown in Fig.
2. The amount of abdominal adipose tissue
increased with age in the wild-type mice. In contrast to the wild-type
mice, the NQO1
/
mice did not accumulate abdominal adipose tissue
with age. This resulted in a significant reduction of abdominal adipose tissue in NQO1
/
mice, especially in the older animals. The 24- and
48-week-old NQO1
/
mice showed a 20 and 38% reduction in abdominal
adipose tissue, as compared with age-matched wild-type controls. The
most significant (p < 0.001) reduction in adipose tissue, between wild-type and NQO1
/
mice, was observed in
72-week-old mice (Figs. 2 and 3). They
showed a 326% reduction in abdominal adipose tissue, as compared with
age-matched wild-type mice.

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Fig. 2.
Abdominal adipose tissue in wild-type and
NQO1 / mice. Twenty-four-, 48-, and 72-week-old wild-type and
NQO1- mice were used. The abdominal adipose tissue was removed by
surgery and weighed. The results are mean ± S.D. of 20 mice in
each group.
|
|
Serum glucose levels of 24-, 48-, and 72-week-old wild-type and
NQO1
/
mice are shown in Table II.
Serum glucose decreased by 11% (p < 0.1), 35%
(p < 0.01), and 36% (p < 0.001) in
24-, 48-, and 72-week-old NQO1
/
mice, when compared with the age matched wild-type mice (Table II). An increase in the pyruvate (37%,
p < 0.01), lactate (18%, p < 0.05),
triglycerides (18%, p < 0.05), and
-hydroxybutyrate (48%, p < 0.007) contents were observed in the serum of 72-week-old NQO
/
mice over the age-matched controls (Table II). Similar results were also observed with mice fasted for 16 h. The concentration of serum glucose decreased by
20% (p < 0.01) and triglycerides showed an increase
by 22% (p < 0.05) in the 72-week-old NQO1
/
mice
as compared with the age-matched wild-type mice. These changes were
more or less similar to that observed when mice were fed ad
libitum. However, the serum
-hydroxybutyrate levels showed
significantly higher differences in fasted mice as compared with mice
fed ad libitum. The NQO1
/
mice showed a 69% increase
(p < 0.005) in the 72-week-old NQO1
/
when compared
with the wild-type mice after 16 h of fasting (Table II). The
NQO1
/
mice livers also demonstrated a marked increase in the
concentration of metabolites such as glucose (105%, p < 0.01), lactate (48%, p < 0.005), pyruvate (79%,
p < 0.005), and triglycerides (180% p < 0.005) as compared with wild-type mice (Table
III). However, liver glycogen reserve of
NQO
/
mice was decreased by 27% (p < 0.025) as
compared with the wild-type mice (Table III). Among the hormones that
regulate metabolism, no significant alterations were observed in
blood-insulin concentrations between NQO1
/
and wild-type mice
(Table IV). However, glucagon levels exhibited a 30% increase (p < 0.05) in the NQO1
/
mice as compared with wild-type mice (Table IV). The insulin tolerance
test in the 16-h fasted wild-type mice showed that the blood glucose
levels in these mice decreased by about 18, 28, and 32% in 20, 40, and 60 min, respectively, after insulin was administered to the animals. In
the NQO1
/
mice the decrease of blood glucose was 6, 9, and 17% at
20 min, 40 and 60 min after administration of insulin (Table V).
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Table II
Concentration of various metabolites in serum of wild-type and
NQO1 / mice (mg/dL)
Mean ± S.D. of six mice. The significance p values are
shown in parentheses.
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Table III
Metabolite levels in livers of 48-week-old wild-type and NQO1 /
mice
Data are mean ± S.D. of six mice. The significance p
values are shown in parentheses.
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Table IV
Serum levels of insulin and glucagon in 48-week-old wild-type and
NQO1 / mice
Data are mean ± S.D. of six mice. The significance p
values are shown in parentheses.
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Table V
Insulin tolerance test in wild-type and NQO1 / mice
Values expressed as percent decrease in blood glucose concentration
after insulin administration from the fasting glucose levels. Values
are mean ± S.D. of five mice. The significance p
values are shown in parentheses.
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|
The amounts of NAD, NADP, NADH, and NADPH and the ratios of NADH/NAD
and NADPH/NADP in the liver and kidney tissues of wild type and
NQO1
/
mice are shown in Table VI for
24- and Table VII for 48-week-old
mice. Seventy-two-week-old mice also showed similar changes as shown
for 48-week-old mice (data are not shown). There was a significant
decrease in the levels of NAD, NADP, NADH, and NADPH in the liver of
24-, 48-, and 72-week-old NQO1
/
mice, as compared with the
age-matched wild-type mice (Tables VI and VII). At 24, 48, and 72 weeks
of age, NADH/NAD and NADPH/NADP ratios were significantly higher in the
liver of NQO1
/
mice, as compared with the wild-type mice. Similar
changes were also observed in the kidney of NQO1
/
mice, as compared
with wild-type mice. However, the magnitude of changes in pyridine
nucleotide levels and NAD(P)H:NAD(P) ratio were less significant than
liver.
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Table VI
NAD(P)H:NAD(P) ratio in liver and kidney tissues of 24-week-old
wild-type and NQO1 / mice
Mean ± S.D. of six mice. The significance p values are
shown in parentheses.
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Table VII
NAD(P)H:NAD(P) ratio in liver and kidney tissues of 48-week-old
wild-type and NQO1 / mice
Mean ± S.D. of six mice. The significance p values are
shown in parentheses. Similar data were observed at 72 weeks.
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|
The above results are shown for male mice. The pattern of alterations
in abdominal adipose tissue, NAD(P)H:NAD(P) ratios, and levels of
glucose, pyruvic acid, lactic acid, and
-hydroxybutyrate observed in
male mice were also observed in female mice (data not shown). However,
the alterations were of lower magnitude in the female NQO1
/
mice.
 |
DISCUSSION |
The NAD(P)H:quinone oxidoreductases are enzymes that are present
in all tissues (1-3). In humans, genetic evidence indicates that
different forms of NQOs are encoded by four gene loci (1-3). Two of
these gene loci (NQO1 and NQO2) encode proteins that constitute ~85%
of the total cellular NQO activity (1). Among all the NQOs known so
far, cytosolic NQO1 is highly abundant and is the most extensively
studied enzyme (1). In the present report, NQO1
/
mice were used to
examine the in vivo role of NQO1.
NQO1
/
mice showed significant decreases in total body weight and
abdominal adipose tissue, as compared with the wild-type mice. The
decrease in abdominal adipose tissue was more significant in older mice
than younger mice. This obvious difference in older (72 weeks) animals
was due to accumulation of abdominal adipose tissue in the wild-type
mice and lower (or no) accumulation of adipose tissue in the aging
NQO1
/
mice. The possible reasons for the decreased abdominal
adipose tissue may be inefficient fat synthesis, ineffective fat
deposition, and/or degradation of adipose tissue.
A decrease in blood glucose and increase in blood and liver lactic and
pyruvic acid indicated that glucose (gluconeogenesis) and possibly
fatty acid synthesis are decreased in NQO1
/
mice as compared with
wild-type mice. The lactic and pyruvic acid are two key substrates for
gluconeogenesis, a metabolic pathway to synthesize glucose (20). Their
accumulation in blood and liver strongly indicated that glucose
synthesis is down-regulated in NQO1
/
mice (20). The lower levels of
blood glucose and higher levels of serum glucagon in NQO1
/
mice
presumably stimulated glycogenolysis in the livers of NQO1
/
mice
leading to increased production of glucose due to breakdown of glycogen
to meet with the energy requirements of the liver. This is supported by
an increase in glucose and decrease in glycogen of liver in NQO1
/
mice as compared with wild-type mice. The increase in glucagon is also
known to stimulate gluconeogenesis (21). However, this may not be the
case in NQO1
/
mice that demonstrated accumulation of pyruvate and
lactate in liver, two substrates for gluconeogenesis. Therefore, it is
likely that most of the glucose in the liver came from the breakdown of
glycogen reserves rather than gluconeogenesis. It is surprising though
that there is sufficient glucose in the liver, yet the serum glucose is
low and the circulating insulin concentration is also unchanged in
NQO1
/
mice. It is possible that NQO1
/
mice liver requires
glucose as its major energy source due to down-regulation of other
metabolic processes leading to increased requirement of glucose in the
livers of NQO1
/
mice. However, the possibility of inefficient
glucose transport from the liver in NQO1
/
mice cannot be ruled out.
The insulin tolerance test demonstrated that NQO1
/
mice are insulin
resistant as compared with the wild-type mice. However, the insulin
resistance did not appear to be of severe form as observed in the case
of lipodystrophic mice (22). We believe that high triglyceride content
in the peripheral blood of the NQO1
/
mice is one of the factors
causing insulin resistance in NQO1
/
mice.
The increase in serum and liver triglycerides in NQO1
/
mice when
compared with wild-type mice may suggest that the breakdown of
accumulated fat also contributed to lower abdominal adipose tissue in
the NQO1
/
mice. An increase in
-hydroxybutyrate in NQO1
/
mice also supported breakdown of adipose tissue. It is known that
-hydroxybutyrate is synthesized from fatty acids generated after the
breakdown of adipose tissue and serves as alternative source of energy
in tissues like brain (20, 23). The hypoglycemic condition in NQO1
/
mice presumably activated alternative pathways of energy generation in
NQO1
/
mice that led to increase in
-hydroxybutyrate production.
At this time, however, the role of inefficient dietary fat deposition
in the adipocytes of NQO1
/
mice in addition to down-regulation of
glucose and fat synthesis and increased fat breakdown remains unknown.
The studies on metabolic precursors and substrates have provided
sufficient evidence that glucose and fat metabolic pathways are altered
in NQO1
/
mice as compared with wild-type mice. It is known that a
physiologically favorable equilibrium between the reduced and oxidized
forms of NAD(P) is crucial for many of the enzymatic reactions that
catalyze reactions leading to the synthesis of pyridine nucleotides,
glucose, and adipose tissue (20). Any alteration in the intracellular
levels of NAD(P)H and NAD(P) is known to affect the pentose phosphate
pathway that generates pentoses for synthesis of pyridine nucleotides,
gluconeogenesis, and fatty acid metabolism (20). In the present
studies, NQO1
/
mice showed decreased pyridine nucleotide levels but
higher ratios of NADPH:NADP and NADH:NAD. NQO1 is known to utilize
NAD(P)H as an electron donor, which generates NAD(P) (1-3). Therefore,
the loss of NQO1 in NQO1
/
mice resulted in the accumulation of
NAD(P)H and alterations in the intracellular redox status. The
accumulation of NAD(P)H presumably inhibited the pentose phosphate
pathway leading to lower levels of pyridine nucleotides in NQO1
/
mice, as compared with wild-type mice. The lower levels of pyridine nucleotides may have contributed to the decrease in gluconeogenesis and
fatty acid metabolism (20). Therefore, an altered intracellular redox
state, caused by higher ratios of NAD(P)H:NAD(P) may be a major factor
that contributed to the down-regulation of gluconeogenesis and adipose
synthesis in NQO1
/
mice.
These results raise an interesting question regarding the endogenous
substrate(s) for NQO1, utilizing NAD(P)H?. Earlier studies have
suggested that NQO1 reduces CoQ and protects membranes from oxidative
damage (24). There may be other cellular molecules that serve as
substrates for NQO1 but remain unknown. There are several proteins that
contain nitrotyrosines (25) and many proteins contain quinones as
cofactors (26). It is possible that NQO1 catalyzes the reduction of
nitrotyrosines or quinone cofactors, thus affecting the structure,
function, and/or stability of these proteins. Future experiments should
reveal more information on the endogenous substrates for NQO1. The
possibility that NQO1 participates in pathways such as the citric acid
and pentose phosphate pathways, as shown for the bacterial malate
oxidoreductase, cannot be ruled out (27).
A model is depicted in Fig. 4 to describe
the role of NQO1 in the regulation of intracellular redox status and
gluconeogenesis. NQO1 utilizes NAD(P)H as an electron donor and
catalyzes the two-electron reduction of known/unknown endogenous
substances and quinones (1-3). This reaction generates oxidized
NAD(P), which maintains a balance between NAD(P)H and NAD(P). This
balance, between the reduced and oxidized form of NAD(P), contributes
to the intracellular redox status that is optimal for pyridine
nucleotides, glucose synthesis, and fatty acid metabolism. Excess
glucose and fatty acids, through metabolic pathways, are converted to
triglycerides that accumulate in the abdominal adipose tissue.
Disruption of the NQO1 gene leads to the loss of NQO1 protein, a
decrease in the concentration of all pyridines (NAD, NADH, NADP, and
NADPH) and accumulation of NADH and NADPH. This leads to an increased intracellular NAD(P)H:NAD(P) ratio that results in an altered intracellular redox status. These changes decrease pyridine nucleotide synthesis, gluconeogenesis, and fatty acid metabolism, resulting in
lower quantities of abdominal adipose tissue. The lower blood glucose
levels may also stimulate degradation of accumulated adipose tissues to
meet the demands of various tissues, including the brain.

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Fig. 4.
A model to demonstrate the role of NQO1 in
regulation of intracellular redox state and gluconeogenesis.
R, substrate(s) for NQO1.
|
|
In conclusion, we have demonstrated that NQO1 plays an important role
in regulating the intracellular redox levels by oxidizing NAD(P)H. The
loss of NQO1 leads to alterations in pyridine and intracellular redox
status. This decreases gluconeogenesis, fatty acid metabolism, and
significantly reduces the amount of abdominal adipose tissue.
 |
ACKNOWLEDGEMENTS |
We are thankful to our colleagues for
discussion. Technical help provided by J. Hariharan for HPLC analysis
is greatly appreciated.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant RO1 ES07943.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.
Contributed equally to the results of this paper.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.:
713-798-7691; Fax: 713-798-3145; E-mail: ajaiswal@bcm.tmc.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M101053200
 |
ABBREVIATIONS |
The abbreviations used are:
NQO1, first
cytosolic form of NAD(P)H:quinone oxidoreductase also known as DT
diaphorase;
NAD(P)H:quinone acceptor oxidoreductase, quinone reductase;
NQO2, a second cytosolic form of NAD(P)H:quinone oxidoreductases;
NAD(P), NAD and NADP combined;
NAD(P)H, NADH and NAD(P)H combined;
HPLC, high performance liquid chromatography.
 |
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