Endocrinology Vol. 139, No. 8 3404-3408
Copyright © 1998 by The Endocrine Society
Effect of Epalrestat, an Aldose Reductase Inhibitor, on the Generation of Oxygen-Derived Free Radicals in Neutrophils from Streptozotocin-Induced Diabetic Rats1
Koji Kashima,
Noriyuki Sato2,
Ken Sato,
Hiroyuki Shimizu and
Masatomo Mori
First Department of Internal Medicine, Gunma University School of
Medicine, 3–39-22, Maebashi Gunma, 371, Japan
Address all correspondence and requests for reprints to: Noriyuki Sato, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-22, Showa, Maebashi, Gunma, 371, Japan. E-mail: satonori{at}sb.gunma-u.sc.jp
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Abstract
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Neutrophil function is impaired by a known mechanism in diabetic
patients, thus increasing susceptibility to infections. We studied the
effect of epalrestat, an aldose reductase inhibitor, on the generation
of oxygen-derived free radicals and cytosolic sorbitol concentration in
neutrophils from streptozotocin-induced diabetic rats. There were four
groups: treated and untreated control and diabetic rats. Treated groups
were given 0.075% epalrestat in their diet for 4 weeks from the
induction of diabetes and were untreated for the subsequent 4 weeks.
Oxygen radicals were measured as chemiluminescence amplified by a
luciferin analog [Cypridina luciferin analog-dependent
chemiluminescence (CLA-DCL), which is dependent on
O2- generation] and luminol (L)-DCL, which is
highly dependent on OCl- generation) in response to
formyl-methonyl-leucyl-phenylalanine. Diabetes resulted in a
significant decrease in CLA/L-DCL and a significant increase in
sorbitol (P < 0.01); there was a negative
correlation between sorbitol and CLA-DCL (P <
0.05) in diabetic groups. The 4-week treatment with epalrestat in the
diabetic group completely prevented the increase in sorbitol and
partially improved the CLA-DCL, although L-DCL was not significantly
affected. After 4 weeks off treatment, CLA-DCL decreased and sorbitol
increased. Treatment had no effect on serum insulin or glucose
concentration. We conclude that an increase in sorbitol in neutrophils
causes, in part, an impaired generation of
O2-. Epalrestat improves the impaired
O2- generation by preventing the sorbitol
increase in streptozotocin-induced diabetic rats.
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Introduction
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NEUTROPHILS play a critical role in the
host defense mechanism against various bacterial infections, and it is
suggested that impaired neutrophil functions (e.g.
chemotaxis, phagocytosis, and bactericidal functions) cause the
susceptibility to infections in diabetic patients (1, 2). Recently, we
(3, 4, 5, 6) and others (7, 8) have demonstrated an impaired production of
oxygen-derived free radicals [e.g. superoxide anion
(O2-) and hydrogen
peroxide-myeloperoxidase-halide
(H2O2-MPO-Cl-) antimicrobial
system] by neutrophils from poorly controlled diabetic patients and
streptozotocin (STZ)-induced diabetic rats. Accordingly, drugs
(e.g. granulocyte-colony stimulating factor) which improve
impaired neutrophils function may prevent or decrease susceptibility to
infection in diabetic patients (9, 10).
One of the major causes of diabetic complication is hyperglycemia, but
the exact mechanism of its detrimental effect is not clear. One
possible mechanism is the accumulation of sorbitol in cells, caused by
activity of the aldose reductase in the polyol pathway. When the
intracellular glucose level is increased in response to an elevation in
plasma glucose level, glucose is metabolized by aldose reductase into
sorbitol, with the reduction of hydronicotinamide adenine dinucleotide
phosphate (NADPH). Limited evidence suggests that one mechanism by
which diabetic state inhibits neutrophil bactericidal function may be
an increase in polyol pathway activity (2). In this study, we have
investigated the effect of epalrestat (5-[1Z,
2E)-2-methyl-3-phenylpropenylidene]-4-oxo-2-thioxo-3-thiazolindineacetic
acid), an aldose reductase inhibitor, on the generation of
oxygen-derived free radicals and cytosolic sorbitol concentration in
neutrophils from STZ-induced diabetic rats. To measure oxygen-derived
free radicals in neutrophils, we employed a Cypridina
luciferin analog (CLA;
2-methyl-6-phenyl-3,7-dihydro-imidazo[1,2-a]-pyrazin-3-one) and
luminol (L; 5-amino-2,3-dihydro-1,4-phthalazine-dione) as agents to
amplify the luminescence. A CLA-dependent chemiluminescence (CLA-DCL)
and L-dependent chemiluminescence (L-DCL) are highly dependent upon the
generation of O2- (11), which is the initial
oxygen-derived free radical and a major intermediate in the formation
of H2O2, and a
H2O2-MPO-Cl- system (12),
respectively. By measuring CLA-DCL and L-DCL, we can determine which
part of oxygen-derived free radicals is changed by epalrestat.
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Materials and Methods
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Animals
Male Wistar rats (200–250 g) were maintained in a
temperature-controlled room (23 ± 1 C) with a 14-h light, 10-h
dark cycle. Rats were randomly assigned to four experimental groups
(Fig. 1
): untreated control,
epalrestat-treated control, untreated diabetic, and treated diabetic
(each group n = 8). Diabetes was induced by a single ip injection
of 60 mg/kg BW STZ (Sigma, St. Louis, MO) dissolved in 0.1
M citric buffer (pH 4.2), as described before (9). A rat
was considered diabetic if it had a nonfasting serum glucose level more
than 20 mmol/liter 3 days after STZ injection. All experimental
procedures were approved by the Gunma University animal ethics
committee.
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Figure 1. Protocol of experiment. Rats were randomly
assigned to four experimental groups: untreated control [CONT,
ARI(-)], epalrestat-treated control [CONT, ARI(+)], untreated
diabetic [DM, ARI(-)], and treated diabetic [DM, ARI(+)].
Open and closed bars indicate that rats
feed normal chow and chow with 0.075% epalrestat, respectively.
Diabetes was induced by a single ip injection of 60 mg/kg BW STZ.
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Treatment protocol (Fig. 1
)
Rats had free access to the standard laboratory chow and
drinking water. In epalrestat-treated groups, epalrestat (Ono, Osaka,
Japan) was added in chow (0.075%) for 4 weeks immediately after the
administration of STZ. For the following 4 weeks, rats were fed chow
powder without epalrestat to determine the off-response. Zero, 4, and 8
weeks after the start of the experiment, 2 ml blood was collected from
the jugular vein under pentobarbital anesthesia; 1 ml was used to
measure serum insulin and glucose levels and 1 ml was used for
neutrophil preparation. Serum insulin was measured by RIA (Pharmaceph
Insulin kit, Pharmacia Japan, Tokyo, Japan). The serum glucose level
was measured by an automatic glucose analyzer, using the glucose
oxidase method (YSI Model 23A, Yellow Spring, CA).
Measurements of active-oxygen radical generation and cytosolic
sorbitol concentration in neutrophils
The neutrophils were separated by the method previously
described (10). The obtained cells, consisting of 95–98% neutrophils,
were suspended in Hanks’ buffer (1 x 106
neutrophils/ml). CLA-DCL and L-DCL of prepared neutrophils were
measured by the method previously described (10). Briefly, 50 µl CLA
or L (final concentration: 5 and 50 µmol/liter, respectively) were
added to the sample tube containing 100 µl of the neutrophil
suspension and 1800 µl Hanks’ buffer. After preincubation for 2 min,
the neutrophils were stimulated by 50 µl
formyl-methonyl-leucyl-phenylalanine (Sigma) solution (final
concentration: 100 nmol/liter). Chemiluminescence from neutrophils was
measured by a Luminescence Reader (Aloka, Inc., Model BLP 102, Tokyo,
Japan). The CLA-DCL and L-DCL were assessed with the peak value of
chemiluminescence as kilocounts/min·106 cells. Cytosolic
sorbitol concentration was measured by the luminescence method
(13).
Statistical analysis
Statistical analysis was made with the Newman-Keuls test for
multiple group comparison. Viability of cells, measured by Trypan blue
staining, was more than 97% at both the beginning and end of the
experiments. All values are expressed as mean ± SE.
Experiments were repeated twice with essentially identical results.
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Results
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Changes in serum insulin and glucose levels
At the beginning of the experiment, no significant differences
were observed in serum insulin and glucose levels among any of the
groups (Table 1). STZ injection
significantly decreased the serum insulin level and increased serum
glucose levels in both treated and untreated diabetic groups. Treatment
with epalrestat had no affect on either serum insulin or glucose
levels. Thus, the effect of epalrestat on the following
chemiluminescence and sorbitol studies are not dependent on changes in
serum insulin and glucose levels.
Changes in CLA-DCL and L-DCL
During the experiment, CLA-DCL and L-DCL did not change in either
the treated or untreated control groups (Fig 2). In treated and untreated diabetic
groups, both CLA-DCL and L-DCL were significantly decreased at 4 and 8
weeks; however, the CLA-DCL at 4 weeks in the treated diabetic group
was significantly higher than that in the untreated diabetes group
(Fig. 2, A and B). After 4 weeks off treatment, CLA-DCL was at the same
low level in both diabetic groups. There was no significant difference
in L-DCL between the treated and untreated diabetic groups at either 4
or 8 weeks. This suggests that epalrestat partially improves the
impaired CLA-DCL, but not the impaired L-DCL, in neutrophils from
STZ-induced diabetic rats.
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Figure 2. Effect of epalrestat on CLA-DCL (A) and L-DCL (B)
by neutrophils from normal and STZ-induced diabetic rats. Data show the
mean ± SE (n = 8). *, P <
0.05; **, P < 0.01 (vs. similarly
treated control groups). #, P < 0.05
vs. DM ARI(-) at 4 weeks.
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Changes in cytosolic sorbitol concentration
Cytosolic sorbitol concentration did not change in either the
treated or untreated control groups during the experiment (Fig. 3). In the untreated diabetic group, the
cytosolic sorbitol concentration was increased at 4 weeks. However, in
the treated diabetic group, the induced decrease in cytosolic sorbitol
concentration was prevented. This difference completely disappeared
after 4 weeks off treatment. This suggests that 4 weeks of treatment
with a 0.075% epalrestat diet clearly prevents the STZ-induced
increase in cytosolic sorbitol concentration.
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Figure 3. Effect of epalrestat on cytosolic sorbitol
concentration in neutrophils from normal and STZ-induced diabetic rats.
Data show the mean ± SE (n = 8). *,
P < 0.05 vs. similarly treated
control groups; #, P < 0.05 vs. DM
ARI(-) at 4 weeks.
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Relationship between CLA-DCL and cytosolic sorbitol concentration
in the diabetic group
In the diabetic group, at 4 weeks, there was a negative
correlation between cytosolic sorbitol concentration and CLA-DCL (Fig. 4A). The change in CLA-DCL was
significantly negative, correlated with the change in cytosolic
sorbitol concentration (Fig. 4B). Thus, the CLA-DCL is affected by the
cytosolic sorbitol concentration in the diabetic group. Epalrestat
improves the impaired CLA-DCL by preventing the STZ-induced increase in
cytosolic sorbitol concentration.
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Figure 4. Relationship between cytosolic sorbitol
concentration and CLA-DCL in diabetic group. Open and
closed circles indicate data from epalrestat-treated and
untreated groups, respectively. A, Relationship between cytosolic
sorbitol concentration and CLA-DCL at 4 weeks; the y-axis
shows CLA-DCL. B, relationship between change in cytosolic sorbitol
concentration and change in CLA-DCL during first 4 weeks; the
y-axis shows the changed CLA-DCL.
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Discussion
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The importance of several oxygen-derived free radicals in the
antimicrobial activity of neutrophils has been recognized (14, 15).
Most of the increased oxygen uptake is used to form
O2- by NADPH oxidase, which is a major
intermediate in the formation of H2O2. This
generated H2O2 participates in the
well-established H2O2-MPO-Cl-
system. We measured two markers of oxygen-derived free radicals,
CLA-DCL and L-DCL. CLA-DCL and L-DCL are dependent on the generation of
O2- and on the
H2O2-MPO-Cl- system, respectively.
In this study, both CLA-DCL and L-DCL in response to
formyl-methonyl-leucyl-phenylalanine were decreased in neutrophils from
STZ-induced diabetic rats. These results were consistent with our
previous observation that the generation of
O2- and a
H2O2-MPO-Cl- system were reduced
in poorly controlled diabetic patients (3, 10) and STZ-induced diabetic
rats (4, 9). In the present study, cytosolic sorbitol concentration was
increased in diabetic neutrophils, which is in agreement with the
results of Wilson’s group (2, 16). They have suggested that an excess
glucose is converted to sorbitol after interaction with the
NADPH-requiring enzyme aldose reductase. The impairment in
O2- generation seen in the diabetic
neutrophils is likely to be caused by competition for NADPH after
induction of this pathway. Thus, our chemiluminescence and sorbitol
data support the Wilson group’s suggestion that the impaired
neutrophil O2- generation is caused by the
increase in cytosol sorbitol concentration in diabetic state.
Recently, some groups demonstrated that ponalrestat, another aldose
reductase inhibitor, protects neutrophils from glucose-related
impairment of killing of E. coli in vivo (17) and Candida
albicans in vitro (18). However, the detailed mechanism of
killing was not clear. Our study is the first to demonstrate that
epalrestat improves the impaired neutrophil killing by protecting
O2- generation in vivo. This effect
may be dependent on the prevention of an increase in cytosolic sorbitol
concentration. This assumption is supported by the following: 1) in
this study, epalrestat prevented both the decrease in CLA-DCL and the
increase in cytosolic sorbitol concentration by diabetic neutrophils,
and there was a significant negative correlation between the change in
cytosolic sorbitol concentration and the change in CLA-DCL; 2) when
epalrestat was withdrawn, the differences disappeared; 3) the diabetic
neutrophil has an active aldose reductase activity, which causes an
increase in cytosolic sorbitol concentration (2). In the present study,
we did not measure intracellular NADPH levels in neutrophils. Thus, we
cannot conclude that an increase in cytosolic sorbitol concentration in
neutrophils from diabetic rats reduces intracellular NADPH levels and,
in turn, reduces neutrophil oxygen-derived free radical generation.
It is well known that the production of oxygen-derived free radicals by
neutrophils is a major factor of killing (14, 15), and there is a good
correlation between oxygen-derived free radical generation and
susceptibility to phagocytosis (19). However, it is important to check
whether treatment of epalrestat improves neutrophil killing. Therefore,
we have additionally studied the effect of epalrestat on neutrophil
killing using the microbiological assay technique (17). The
experimental groups were the same as shown in Fig. 1
(treated/untreated control and diabetic groups). The assay was
performed at 4 weeks after the start of experimentation. Unfortunately,
there were not significant differences among groups. We cannot clearly
explain the discrepancy of data from chemiluminescence and above
killing study. One possibility is that the discrepancy may be caused by
the different sensitivities of the two assays. The killing assay is
known to have much intraindividual and interindividual variation. The
other possibility is a difference between epalrestat and ponalrestat.
In our present study, epalrestat prevented the STZ-induced decrease in
CLA-DCL. However, the prevention was partial, and L-DCL was not
affected by epalrestat. Thus, another sorbitol-independent mechanism
participates in the impaired O2- generation by
diabetic neutrophils, and the impaired
H2O2-MPO-Cl- system is not caused
by an increase in cytosolic sorbitol concentration. The mechanism by
which diabetes inhibits neutrophil bactericidal function is undoubtedly
complex. We and others have already suggested that there are at least
two other mechanisms by which diabetes can impair neutrophil function.
First, the inhibition of MPO activity may be mediated by an allosteric
blockade after glycosylation of the enzyme in poorly controlled NIDDM
(3). Second, intracellular messenger systems (e.g. cytosolic
Ca2+ and protein kinase C activity) are altered in diabetic
neutrophils, and impaired neutrophil function may be caused by the
changes in second-messenger systems (20).
Despite the great improvement brought by insulin and antimicrobial
agents, bacterial infection still accounts for an important cause of
morbidity and mortality in diabetic patients (1, 2). This is the first
study demonstrating that epalrestat improves the impaired
O2- generation by preventing the
hyperglycemia-induced increase in sorbitol concentration in
vivo. We suggest that epalrestat may help to prevent the
susceptibility to infection in diabetic patients. Obviously, much work,
including measurement of cytosolic NADPH concentration, and clinical
trial, remains to clarify, in more detail, the mechanism by which
Epalrestat improves the generation of O2- and
to establish the significance of our observations.
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Acknowledgments
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We are very indebted to Dr. Monte A. Greer for examining the
English in the manuscript.
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Footnotes
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1 This study was supported, in part, by Research Grant 96710231 from
the Ministry of Education of Japan. 
2 Present address; Department of Endocrinology, Dokkyo Medical
University, 880, Kobayashi, Mibu, Simotsuga, Tochigi, 321–02,
Japan.
Received December 12, 1997.
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Abstract
Introduction
