Endocrinology Vol. 142, No. 4 1442-1447
Copyright © 2001 by The Endocrine Society
Testosterone Effect on Insulin Content, Messenger Ribonucleic Acid Levels, Promoter Activity, and Secretion in the Rat1
Sumiko Morimoto2,
Cristina Fernandez-Mejia,
Guillermo Romero-Navarro,
Nestor Morales-Peza and
Vicente DÍaz-Sánchez
From the Department of Reproductive Biology, National Institute of
Medical Sciences and Nutrition Salvador Zubiran (S.M., V.D.S.),
Nutritional Genetics Unit (C.F.M., G.R.N.), Biomedical Research
Institute, National University of México, National Institute of
Cancer (N.M.P.), Tlalpan 14000, D.F. México City,
México
Address all correspondence and requests for reprints to: Vicente Díaz-Sánchez, M.D., Departamento de Biología de la Reproducción. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. Vasco de Quiroga 15, Tlalpan 14000, D.F. México City, México. E-mail:
vidisa{at}quetzal.innsz.mx
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Abstract
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Coexistence of hyperinsulinemia and hyperandrogenism in women has been
frequently described. Most of the studies addressing this issue have
focused on the mechanisms by which insulin produces hyperandrogenism.
In the present study, we analyzed the effects of testosterone in
vivo and in vitro upon insulin gene expression
and release in the rat. Our studies demonstrate that testosterone
increases insulin messenger RNA (mRNA) levels in vitro
as well as in vivo. In both prepuberal and intact adult
rats, serum testosterone concentrations were positively correlated with
insulin mRNA levels and insulin concentration in serum. Testosterone
deprivation after gonadectomy decreased both insulin gene expression
and serum insulin concentration. Insulin mRNA levels were partially
restored after 3 days of testosterone administration and serum insulin
was 80% and 27% above baseline values at 5 and 7 days posttreatment.
Primary cultured pancreatic islets treated with the sexual steroid
increased about 80% insulin mRNA, as well as protein, and release. In
transfected islets, testosterone increased the activity of the -410 bp
rat insulin promoter I by 154%. These data demonstrate that
testosterone has a direct effect upon pancreatic islet function by
favoring insulin gene expression and release.
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Introduction
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THE ASSOCIATION between disorders of
carbohydrate metabolism and hyperandrogenism in women has been known
for more than 60 yr (1). Coexistence of insulin resistance
and high circulating androgens in women has been described frequently,
mainly in association with the polycystic ovary syndrome (PCO)
(2). Several studies have demonstrated a striking positive
correlation between hyperandrogenism and hyperinsulinemia in PCO with
relatively mild glucose intolerance (3, 4). Prolonged
testosterone administration to female organisms, which produced
circulating testosterone levels in the male range, resulted in a
significant decrease on insulin-mediated glucose uptake (5, 6). Androgen-mediated insulin resistance may be the result of an
increase in the number of less insulin-sensitive type II skeletal
muscle fibers (7) and an inhibition of muscle glycogen
synthase activity (8). Besides the syndromes of extreme
insulin resistance are commonly associated with hyperandrogenism when
they occur in premenopausal women. Insulin can stimulate ovarian
estrogen, androgen, and progesterone secretion in vitro.
Although some of these actions have been observed at physiological
insulin concentrations, most actions have been observed at high insulin
concentrations (9). Most of the reported actions of
insulin on steroidogenesis have been observed in women with PCO. These
observations suggest that if insulin facilitates ovarian
hyperandrogenism in women, polycystic ovarian changes should promote
ovaries to secrete higher levels of androgens (10). Most
research efforts in this field have been focused on the mechanisms of
insulin that produce hyperandrogenism, and few information is available
on the effects of androgens upon the synthesis and release of
insulin.
The aim of the present study was to assess the effects of testosterone
in vivo and in vitro upon insulin gene
expression, insulin biosynthesis and insulin release in the rat.
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Materials and Methods
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Animals
Four groups of Wistar male rats (Harlan, México City,
México) were formed as follows: A, Prepuberal (21 days old); B,
intact adult (90 days old); C, gonadectomized adults; D, gonadectomized
and restored with testosterone. Gonadectomy was performed under ether
anesthesia. In the group receiving testosterone, 72 h after
surgery, 5 mg of testosterone enanthate was administered by im
injection. After 3, 5, and 7 post testosterone treatments, pancreatic
tissue was isolated.
Serum hormones
Blood samples were collected from ad libitum fed rats
by cardiac puncture under ether terminal anesthesia. Serum was obtained
by centrifugation and stored at -20 C for insulin and testosterone
determination. Insulin and testosterone were measured by specific RIAs
(ICN Biomedicals, Inc., Costa Mesa, CA; Diagnostic Products Corp., Los Angeles, CA).
Adult islet culture
Pancreatic islets were isolated from ad libitum fed
Wistar male rats (200–250 g) injected ip with 0.25 ml of a 6.3%
solution of pentobarbital (Pfizer, Inc., México
City, México). Islets were obtained by the collagenase method
previously reported (11). Islets were suspended in
Rosewell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS
and 400 U/ml penicillin, and 200 mg streptomycin (Life Technologies, Inc., Gaithersburg, MD) (supplemented RPMI) and
distributed equally into 60-mm tissue culture dishes
(Costar, Cambridge, MA). Cultures were incubated at 37 C
in a humidified atmosphere of 5% CO2; and
after 18 h of plating, cells were treated either with
testosterone (1 µg/ml) or vehicle (ethanol, 0.01%). To avoid bias in
the interpretation, all results were normalized to DNA content assessed
by spectrophometric analysis at 260 nm (untreated islets 5.71 ±
0.75 µg; treated 5.86 ± 0.69 µg).
RNA extraction and Northern blot analysis
Total RNA was isolated by homogenizing in Trizol (Life Technologies, Inc., Gaithersburg, MD). For the in
vivo studies, pancreatic tissue was removed by surgery under
terminal ether anesthesia; from each tissue sample, 20 µg total RNA
were used for Northern blot analysis. For the in vitro
studies, 5 µg of total RNA from the cultured islets were used for the
analysis. RNA samples were denatured and electrophoresed on 2.2
M formaldehyde 1% agarose gel. Electrophoresed
samples were then transferred to a nylon membrane (Hybond-N nylon,
Amersham Pharmacia Biotech, Arlington Heights, IL), and
cross-linked in a Stratalinker UV cross-linker 1800
(Stratagene, La Jolla, CA). Blotted membranes were
hybridized with the following probes: 1) The insulin probe was a 306-bp
PstI fragment (374–680) from a human insulin complementary
DNA (American Type Culture Collection, Manassas, VA). 2)
the actin probe was a -410 bp SmaI/HpaII
fragment subcloned in pGEM (12). Hybridization was carried
out at 42 C overnight to complementary DNA probes labeled with
32P by random priming procedure (Roche Molecular Biochemicals, Mannheim, Germany). For intraspecies
hybridization, membranes were washed to high stringency using 0.1
x SSC, 0.1% SDS at 65 C; for cross-hybridization, membranes were
washed in the same solution at 50 C. Membranes were exposed to
Kodak X AR x-ray film (Rochester, NY) at -70 C, using
intensifying screens. Autoradiograms were scanned on an image
densitometer (Eagle eye II, Stratagene).
Plasmids
A construct containing rat I promoter sequences -410 +1 linked
to the CAT reported gene cloned into pFOX vector (13) was
used.
Adenovirus preparation
Virus proliferation was performed according to (14)
in HEK 293 cell line. After 36 h of primary infection, cells were
lysed by freezing/thawing in liquid nitrogen. Virus were purified by
Freon 11 gradient and mature virus were recovered by
ultracentrifugation in cesium chloride gradient (15).
Islets transfection
Islet transfection was performed as reported by Sander et
al. (15). Briefly, 50 adult islets were placed in
12 x 75 mm culture tubes and washed three times with one ml of
Opti-MEM (Life Technologies, Inc.). Plasmid DNA (4 µg)
was diluted in 100 µl Opti-MEM. Four micrograms of polycationic
dendrimers (16) were diluted in 50 µl Opti-MEM, added to
the diluted plasmid DNA, and incubated at room temperature for 15 min.
Replication-deficient adenovirus 5 dI-343 were diluted in 100 µl
Opti-MEM to produce approximately a final concentration of 3.5 x
109 virus particles per ml. The diluted
adenovirus were added to the plasmid DNA/dendrimers and incubated at
room temperature for an additional 15 min period. The
DNA/dendrimers/adenovirus mixture was then added to the islets and
incubated for 30 min at 37 C in a humidified atmosphere of 5%
CO2. Cells were washed twice in supplemented RPMI
and cultured at 37 C in a humidified atmosphere of 5%
CO2 in the presence of testosterone (1 µg/ml)
or vehicle (ethanol, 0.01%).
Choramphenicol acetyl-transferase assay (CAT)
Islets were washed twice in 1 ml HBSS (Life Technologies, Inc.). Cell pellets were disrupted by sonication and 1.5 µg/ml
of cell extract was analyzed for CAT activity in the presence of
acetyl-CoA and C (14) chloramphenicol. Acetylated products
were separated by TLC as reported previously (17).
Insulin content
Adult islets were isolated and cultured as described above.
After 18 h plating, medium was changed. Islets were then incubated
for 3 h in the presence of testosterone (1 µg/ml) or vehicle
(ethanol, 0.01%). Islets were washed in PBS buffer, centrifuged, and
treated with ethanol-acid at 0 C overnight. Insulin content was
analyzed by RIA (ICN Biomedicals) and values were
normalized by protein content (Bio-Rad Laboratories, Inc.,
Richmond, CA). Protein concentrations were 25.34 ± 3.0 µg and
24.47 ± 2.86 µg for the treated and control samples,
respectively.
Insulin secretion
Adult islets were isolated and cultured as described above.
After 18 h isolation, islets were washed twice with secretion
buffer containing 20 mM HEPES, 115 mM NaCl, 5
mM NaHCO3, 4.7 mM KCl,
2.6 mM CaCl2, 1.2 mM
KH2PO4, and 1.2
mM MgSO4 (pH 7.4). Islets were then
incubated for 3 h in the secretion buffer containing either,
testosterone (1 µg/ml) or vehicle (ethanol, 0.01%), in the absence
or presence of 5.5, 11, or 16 mM D-glucose.
Medium was collected and insulin concentration was measured by RIA
(ICN Biomedicals) and normalized by protein content
(Bio-Rad Laboratories, Inc.).
Statistics
Data are presented as mean ± SE. Multiple
comparisons were calculated by one way ANOVA. Individual comparisons
were evaluated by Student’s paired two tailed t test. The
significance level chosen was P < 0.05.
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Results
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In vivo studies
Insulin messenger RNA (mRNA) levels in different endocrine
conditions. Insulin gene expression was analyzed in pancreas of
prepuberal, normal adult, adult gonadectomized, and testosterone
supplemented gonadectomized rats. Our results revealed significant
differences (P 
0.05) among the experimental animal
groups (Fig. 1
): insulin gene expression
in the prepuberal stage was about 15.3 ± 8.2% of the value found
in adult intact male rats. In gonadectomized males, insulin mRNA levels
were decreased to 44.2 ± 5.1% of the values observed in normal
rats. Testosterone replacement after gonadectomy increased insulin
mRNA, although 3 days after treatment the increase produced did not
restore insulin mRNA to the levels observed in the intact adult
animal.
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Figure 1. Insulin gene expression in different endocrine
conditions. PP, Prepuberal; IA, intact adult rats; GNX, gonadectomized;
GNX + T, gonadectomized males supplemented with 5 mg testosterone
enanthate. Pancreatic tissue was collected from fed Wistar male rats.
Insulin and actin mRNA levels were analyzed by Northern blot.
Hybridization signals were quantitated by densitometric scanning, each
sample was standardized to actin. Data are expressed as relative to
that measured in intact adult rats. Each value represents the mean
± SE of six independent experiments. One representative
autoradiogram is also shown. Significance was assessed by one-way ANOVA
(*, P 0.05).
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Serum insulin and testosterone concentrations in different
endocrine condition. We next investigated whether the changes on
insulin gene expression were reflected in serum insulin concentrations.
As can be seen in Fig. 2, serum insulin
concentrations were significantly (P 0.05) lower in
prepuberal rats than in intact adult males. Gonadectomy produced a
decrease in circulating insulin, although this reduction was not
statistically different compared with adult rats (gonadectomized
25.2 ± 4.9 µU/ml; intact adult 29.9 ± 3.6 µU/ml
P > 0.05). As expected, gonadectomized rats
supplemented with pharmacological doses of testosterone exhibited high
levels of the steroidal hormone (Fig. 2B). At 3 days post treatment
serum insulin values were only 67% of those observed in normal adult
rats; however, at 5 and 7 days post treatment insulin concentrations
were 80% and 27% higher of those found in the intact adult group.
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Figure 2. Serum insulin and testosterone concentrations in
different endocrine condition. Blood samples were collected from fed
Wistar male rats (n = 6), insulin and testosterone concentration
in the serum were determined by RIA. PP, Prepuberal; IA, intact adult
rats; GNX, gonadectomized; GNXT3; GNXT5; GNXT7, gonadectomized males
supplemented with 5 mg testosterone enanthate at 3, 5, and 7 days post
treatment. Data are expressed as the mean ± SE of six
independent experiments. (*, P 0.05) compared
with intact adult.
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In vitro studies
Effect of testosterone on insulin mRNA levels in primary cultures
of pancreatic islets. To investigate whether testosterone was able
to directly affect insulin expression, we analyzed the effect of the
steroid in islets obtained from normal adult male rats. As shown in
Fig. 3, 3 h incubation with
testosterone concentrations of 1 µg/ml, increased insulin mRNA levels
by 65 ± 17.5%. In time-course experiments (data not shown), we
found that testosterone produced its maximal effect after 3 h of
treatment. Not significant differences were found with longer
incubation times. To assess whether the stimulatory effect of
testosterone was dependent of its presence in incubation media, we
removed the androgen by serial washes and maintained the islets
in steroid-free culture for another 24 h. After this incubation
period, mRNA levels were analyzed, and the concentration was not
significantly different (45 ± 15%) to that found in cells
maintained in culture for 3 h in the presence of testosterone.
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Figure 3. Effect of testosterone on insulin mRNA levels in
primary cultures of pancreatic islets. Islets were isolated by
collagenase digestion and cultured as described in Materials and
Methods. After 3 h incubation in the presence of
testosterone (1 µg/ml) (T) or vehicle (ethanol [0.01%]) (C), RNA
was extracted and insulin and actin mRNA were analyzed by Northern
blot. Hybridization signals were quantitated by densitometric scanning.
Each sample was standardized to actin. Data are expressed as relative
to that measured in islets without treatment. Each data represents the
mean ± SE of five independent experiments. One
representative autoradiogram is also shown. Significance was assessed
by Student’s unpaired two-tailed t test. (*,
P 0.05).
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Effect of testosterone on insulin promoter activity. A
construct containing rat I promoter sequences -410 +1 linked to the
CAT reporter gene cloned into pFOX vector driving the CAT was
transfected in primary cultures of adult islets. An increase of
154 ± 47% of insulin promoter activity as measured by CAT enzyme
activity (Fig. 4) was observed after
21 h of treatment with testosterone doses of 1 µg/ml. No effect
was observed on the empty pFOX-CAT1 vector (control = 100%,
testosterone = 95 ± 9.3%)
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Figure 4. Effect of testosterone on insulin promoter
activity in primary cultures of pancreatic islets. Adult islets were
transfected with pFOXCAT1 containing -410 to +1 bp of rat I insulin
promoter, as described in Materials and Methods. The
islets were then incubated in medium with testosterone (1 µg/ml) (T)
or vehicle (ethanol, 0.01%) (C) for approximately 21 h. CAT
activity was then assayed in 1.5 µg protein of the cell extracts and
expressed relative to that measured in cells incubated with vehicle.
One representative autoradiogram is also shown. Each value represents
the mean ± SE of three experiments. Significance was
assessed by Student’s unpaired two-tailed t test. (*,
P 0.05).
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Effect of testosterone on insulin content. We also determined
the effect of testosterone on insulin content in primary cultures of
pancreatic islets. As shown in Fig. 5, 3
h incubation with testosterone concentrations of 1 µg/ml
increased insulin content by 60.5 + 9%.
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Figure 5. Insulin content in primary cultures of pancreatic
islets. Islets were isolated by collagenase digestion. Islets were
cultured for 3 h in the presence of testosterone (1 µg/ml) (T)
or vehicle (ethanol, 0.01%) (C). Insulin was extracted by ethanol-acid
and concentration determined by RIA. Data are expressed as the
mean ± SE of five independent experiments.
Significance was assessed by Student’s unpaired two-tailed
t test. (*, P 0.05).
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Effect of testosterone on insulin release. We analyzed the
effect of testosterone on insulin release at different glucose
concentrations. As can be seen in Fig. 6, 3 h of treatment with 1 µg/ml of testosterone increased insulin
release in either absence or presence of glucose.
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Figure 6. Effect of testosterone on insulin secretion in
primary cultures of pancreatic islets. Islets were cultured for 3
h in secretion buffer containing the indicated concentrations of
glucose in either the presence of testosterone (1 µg/ml)
(dashed bars) or vehicle (ethanol, 0.01%) (black
bars). The insulin secreted to the medium was collected and
determined by RIA. Data are expressed as the mean ±
SE of five independent experiments. Multiple comparisons
were evaluated using one-way ANOVA. (*, P 0.05).
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Discussion
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To our knowledge, this study represents the first report on the
effects of testosterone on insulin gene expression. Our results
demonstrate that testosterone increases insulin mRNA levels in
vitro as well as in vivo. The stimulating effect of
testosterone is also observed on insulin promoter activity, content and
release, as demonstrated in our in vitro studies. In
physiological conditions such as those presented in prepuberal and in
intact adult rats, circulating levels of testosterone were in
correspondence with both, insulin gene expression in pancreas and
concentration in serum. The relationship between testosterone and
insulin expression was also observed in gonadectomized animals;
however, the parallelism between serum concentrations of testosterone
and insulin production was not reflected in the same extent. The strong
reduction in testosterone levels produced by gonadectomy decreased both
insulin gene expression and insulin concentrations in serum, although
these reductions were not as pronounced as the circulating steroidal
levels. The high levels of testosterone produced in gonadectomized rats
treated with pharmacological doses of the hormone, restored partially
insulin mRNA levels and did not further increase serum insulin
concentration after 3 days of treatment. This partial effect might be
due to other metabolic and hormonal changes produced as result of
gonadectomy (18, 19); it is also conceivable that
compensatory mechanisms that maintain serum insulin homeostasis such as
insulin uptake and degradation account for this effect; furthermore, it
has been demonstrated that androgen receptors interact with insulin
degrading enzyme (20). Interestingly, insulin serum
concentrations were restored and even enhanced at 5d and 7d
posttreatment. These data suggest that a sustained high concentrations
of testosterone might enhance insulin release as observed in women with
hyperandrogenism.
Testosterone effects on gene expression are known to be mediated
through the activation of its nuclear receptors (21). We
have previously demonstrated the presence of the androgen receptor in
pancreas (22). The fact that testosterone was capable to
increase insulin I promoter activity and that the effect of
testosterone on insulin mRNA levels were rapidly achieved suggested
that the steroid should have a direct effect on insulin gene
transcription. No putative androgen response elements appeared to be
present in the insulin gene; however, it is possible that other
transcriptional mechanisms as androgen receptor interaction with basal
transcription machinery (23) or interaction with other
coactivators that bind the androgen receptor (24, 25)
could be involved in the mechanism by which testosterone increases
insulin promoter activity.
The studies presented here also demonstrate that cultured islets
treated with testosterone increased insulin content. A stimulatory
effect of testosterone on insulin content has also been reported by
Fang et al. (26) in interleukin-1-ß treated
islets; these authors have also found that the inhibitory effect of
IL-1-ß on insulin release could be reversed by testosterone
(26). Our studies show that testosterone alone was able to
increase insulin release. These data are in contrast to those obtained
by Nielsen in organ culture (27), who found that the
treatment with testosterone for 14 days did not significantly affect
insulin secretion, the discrepancy may be related to the different
length of culture conditions. The fact that testosterone augmented
insulin release in response to glucose below the stimulatory
concentrations, suggests that the steroid does not modulate the
regulatory pathway of insulin secretion. Testosterone has been shown to
induce calcium influx via nongenomic surface receptors
(28, 29, 30), whether the stimulatory effect of testosterone
on insulin secretion is related to its nongenomic effects on
intracellular calcium concentration (28, 29, 30), glucose
metabolism, or changes in second messengers, will await further
investigations.
Theories of the pathophysiology of PCO have implicated primary defects
in hypothalamic-pituitary function, ovarian activity, and peripheral
insulin action (31). The present study demonstrates that
androgens have a direct effect upon the ß cell, promoting the
expression and release of insulin. This not previously disclosed
mechanism of hyperinsulinemia adds support to the current theory of the
multifactorial causes of PCO. This direct effect of androgens on
insulin production, is another reason to treat the hyperandrogenism in
women with PCO. Effective antiandrogen treatment not only could improve
the phenotypic and metabolic effects of androgens, but also could
contribute to the amelioration of hyperinsulinemia.
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Acknowledgments
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The authors are grateful to Dr. Octavio Villanueva for his help
in animal care. We thank Fernanda Tenorio and Renata Rivera, Alberto
Rojas Ochoa, and Christian Guerra Araiza for technical assistance. We
would like to thank Dr. Ignacio Camacho-Arroyo for his critical reading
of this manuscript. The construct containing rat I promoter -410 +1
FOXCAT was kindly provided by M. German (University of Californina San
Francisco, San Francisco, CA).
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Footnotes
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1 This work was supported by the Consejo Nacional de Ciencia y
Tecnología (CONACyT) Grants 4025-M 9403 (to C.F.M.), 28916-M
(to V.D.S.). 
2 Recipient of a scholarship granted by the Consejo Nacional de
Ciencia y Tecnología (CONACyT, México).
Received July 28, 2000.
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A. Morales, S. Morimoto, L. Diaz, G. Robles, and V. Diaz-Sanchez
Endocrine gland-derived vascular endothelial growth factor in rat pancreas: genetic expression and testosterone regulation
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Top
Abstract
Introduction
