Eur J Nutr (2013) 52:347359

DOI 10.1007/s00394-012-0342-4

ORIGINAL CONTRIBUTION

Alterations in peripheral purinergic and muscarinic signaling

of rat bladder after long-term fructose-induced metabolic
syndrome
Shiu-Dong Chung Chiang-Ting Chien
Hong-Jeng Yu

Received: 16 October 2011 / Accepted: 5 March 2012 / Published online: 18 March 2012
Springer-Verlag 2012

Abstract
Purpose We explored the pathophysiologic mechanisms
of long-term fructose-induced lower urinary tract symptoms (LUTS) in rats.
Methods Male Wistar rats were fed with fructose for 3 or
6 months. Biochemical and transcystometric parameters
were compared between fructose-fed and age-matched
normal-diet rats. Pelvic nerve and external urethral
sphincter-electromyogram activity recordings were performed to investigate fructose effects on neural control of
bladders. Mitochondrial structure, ATP and acetylcholine
content and purinergic and muscarinic cholinergic receptors were examined. Cytosolic cytochrome C staining by
Western blot and immunocytochemistry for mitochondrial
injury and PGP 9.5 stain for nerve density were also
determined.
Results The fructose-fed rats with higher plasma triglyceride, LDL and fasting glucose levels displayed LUTS
S.-D. Chung
Department of Urology, Far-Eastern Memorial Hospital,
New Taipei City, Taiwan
S.-D. Chung
C.-T. Chien
H.-J. Yu
Graduate Institution of Clinical Medicine,
National Taiwan University College of Medicine,
Taipei, Taiwan
C.-T. Chien
Departments of Medical Research, National Taiwan University
Hospital and National Taiwan University College of Medicine,
Taipei, Taiwan
H.-J. Yu (&)
Department of Urology, National Taiwan University Hospital
and National Taiwan University College of Medicine,
7 Chung-Shan South Road, Taipei, Taiwan
e-mail: [email protected]

with increased frequency and suppressed voiding contractile amplitude in phase 1 and phase 2 duration versus
normal-diet control. Fructose feeding altered the firing
types in pelvic afferent and efferent nerves and external
urethral sphincter-electromyogram activity. Increased
mast cell number, disrupted and swollen mitochondria,
increased cytosolic cytochrome C stain and expression and
decreased nerve density in bladder smooth muscle layers
appeared in the fructose-fed rats. Fructose feeding also
significantly reduced ATP and acetylcholine content and
enhanced protein expression of postsynaptic P2X1, P2X2
and P2X3 purinergic receptors and M2 and M3 muscarinic
cholinergic receptors expression in the smooth muscles of
urinary bladder.
Conclusion Long-term fructose feeding induced neuropathy and myopathy in the urinary bladders. Impaired
mitochondrial integrity, reduced nerve density, ATP and
acetylcholine content and upregulation of purinergic and
muscarinic cholinergic receptors expression may contribute
to the bladder dysfunction of fructose-fed animals.
Keywords Urinary bladder
Metabolic syndrome

Male rats
Purinergic receptor
Muscarinic receptor

Introduction
Metabolic syndrome and diabetes are known risk factors
for the development of lower urinary tract symptoms
(LUTS) including a high prevalence of overactive bladder
[14]. The increased high-fructose consumption results in
obesity and metabolic syndrome [5, 6], which may contribute to vesicular neuropathy. In patients, there were
linear associations between storage/voiding LUTS (frequency and incomplete emptying) and increased abdominal

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fat mass, plasma glucose and low-density lipoprotein

cholesterol (components of the metabolic syndrome) [7].
Alterations in these biochemical parameters may lead to
oxidative stress and mitochondria injury in neural, vascular
and muscular dysfunction [810]. In the urinary bladder,
ATP-purinergic and acetylcholine-muscarinic cholinergic
signaling associated with coordinated external urethral
sphincter activity are required for performing an efficient
voiding contraction [1113]. Several studies have demonstrated that the muscarinic cholinergic and purinergic signaling were altered in detrusor muscle of variable urologic
diseases [14, 15].
The fructose-fed rat model has been well developed with
a very similar metabolic profile to the human condition
[16]. Furthermore, a bladder dysfunction with bladder
overactivity in vivo and an alteration in contractile activity
to KCl, muscarinic cholinergic agonist or ATP in vitro was
noted in the urinary bladder after long-term fructose
feeding [16]. Therapeutic potential for using muscarinic
cholinergic and/or purinergic antagonist has been frequently discussed in the diseases of the urinary tract [17
19]. Because up to 40 % of the neural responses were
purinergic in the pathological situations including interstitial cystitis, outflow obstruction, idiopathic detrusor
instability and neurogenic bladder, P2X receptor antagonists are being explored as therapeutic agents [17]. The
therapeutic potential of the cholinergic system is also well
established in the urinary tract diseases and using the
muscarinic antagonists being the primary treatment for
patients with the overactive bladder [18, 19].
Therefore, in this study, we attempt to explore the neural
mechanisms underlying bladder dysfunction by the longterm effects of metabolic syndrome on male rat urinary
bladder function. The bladder in male rat fed with highfructose-containing diet for 3 and 6 months will be compared to age-matched normal controls. In addition to biochemical, physiologic, pathologic and transcystometric
parameters, we investigated and analyzed the neurophysiologic recordings of pelvic afferent and efferent nerve
activity and external urethral sphincter-electromyogram,
mitochondrial structure, ATP and acetylcholine content,
purinergic (P2X1, P2X2 and P2X3) and muscarinic cholinergic receptor (M2 and M3) expression of the urinary
bladder and immunostaining with PGP 9.5 for examination
of the nerve density in the urinary bladder.

Methods and procedures

Experimental animals
Twenty-eight male Wistar rats were housed at the Experimental Animal Center, National Taiwan University, at a

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constant temperature and with a consistent light cycle (light

from 07:00 to 18:00 oclock). Fructose-fed animals were
fed a fructose-rich diet (60 % fructose diet, Harlan Teklad),
whereas control animals received standard rat chow for 3
or 6 months, respectively. The rats were divided into four
groups as following characterization: 3 months of fructosefed rats with LUTS (n = 8); 6 months of fructose-fed rats
with LUTS (n = 8), and age-matched control groups for 3
or 6 months of standard rat chow (n = 6 each). All surgical
and experimental procedures were approved by National
Taiwan University College of Medicine and College of
Public Health Institutional Animal Care and Use Committee and are in accordance with the guidelines of the
National Science Council of Republic of China (NSC
1997). All efforts were made to minimize animal suffering
and the number of animals throughout the experiment.
Baseline parameters
After 3 or 6 months of treatment, the metabolic characteristics and baseline of micturition parameters were
measured in all animals. All animals were anesthetized
with subcutaneous urethane (1.2 g/kg), which is known to
anesthetize the animals permit full reflex bladder contractions [12]. Body temperature of the animal was kept at
36.537 C by an infrared light and evaluated by a rectal
thermometer. At the end of each experiment, the animals
were killed by an intravenous KCl injection. Blood samples
were collected immediately to measure blood glucose (One
Touch II; LIFESCAN, Milpitas, CA, USA), cholesterols,
low-density lipoprotein and triglycerides [16]. Hepatic
steatosis, an index of metabolic syndrome, was assessed
using oil red O staining. Oil red O staining was performed
to evaluate the accumulation of fat droplets in hepatocytes
in frozen liver sections from the fructose-feeding rats. The
bladder was removed and one part of the bladder was fixed
in 4 % buffered formalin for morphological staining. Part
of the samples was stored at -70 C for postsynaptic
receptor protein expression by Western blot analysis. Part
of samples was OCT embedded for nerve distribution by
PGP 9.5 stain.
Cystometric findings
We applied a transcystometric model to evaluate the micturition response in the bladder after fructose feeding [16].
The method has been well established in our laboratory
[12, 13]. The bladder catheter was connected via a T-tube
to a pressure transducer (P23 1D, Gould-Statham, Quincy,
USA), and the intravesical pressure (IVP) was recorded
continuously in an ADI system (Power-Lab/16S, ADI
Instruments, Pty., Ltd., Castle Hill, Australia). The following parameters of bladder responsiveness were

Eur J Nutr (2013) 52:347359

measured: intercontraction interval (ICI, the time lag

between two micturition cycles identified with active
contractions ([15 mmHg), baseline bladder pressure (BP),
threshold pressure (TP) and peak intravesical pressure
(pIVP).
Recording of pelvic afferent and efferent nerve activity
The electrophysiological technique used to record pelvic
afferent and efferent nerve activity was described previously [12, 13]. In brief, with the help of a stereoscopic
dissecting microscope (Olympus, SZ-STU2), pelvic afferent and efferent nerve activities were recorded simultaneously from two pelvic nerves dissected from the surface
of the bladder. Nerve fiber with pelvic afferent activity was
confirmed by its ability to show increased activity in
response to small increments of IVP by saline infusion via
T-tube. Nerve fiber with pelvic efferent nerve activity had
minimal activity until a threshold volume/pressure in the
bladder was reached, which produced a bursting discharge
causing a micturition contraction.
Recording of extraurethral sphincter-electromyogram
activity
We used epoxy-coated stainless steel wire (50 lm,
M. T. Giken Co., Tokyo, Japan) as an electrode for determining extraurethral sphincter-electromyogram activity
[12]. In brief, a 30-gauge needle with a hooked extraurethral
sphincter-electromyogram electrode was inserted into the
sphincter approximately 510 mm lateral to the urethra.
Extraurethral sphincter-electromyogram signals were
amplified and passed through a discriminator/rate meter.
Pelvic afferent and efferent nerve activities, extraurethral
sphincter-electromyogram activity and cystrometrogram were simultaneously recorded by an ADI system
(Power-Lab/16S, ADI Instruments, Pty., Ltd., Castle Hill,
Australia).
ATP and acetylcholine content assay
The portion of the bladder above the ureteral orifices was
harvested as the bladder body. Under a dissecting microscope (Olympus SZ61), we obtained the muscle layers
from the bladder body wall by stripping off the urothelium
and mucosa with sharp tweezers (DUMONT, Switzerland).
To avoid cross-contamination by these two layers, the
proficiency of separation was assessed histologically as
described previously [11]. We further analyzed the ATP
and acetylcholine content and postsynaptic receptor
expression in the isolated smooth muscle layers. The
smooth muscle layers were collected and stored at -70 C
for ATP and acetylcholine content analysis. The method

349

for measurement of bladder acetylcholine content was used

by a commercial kit (Choline/Acetylcholine Quantification
Kit, K615-100, BioVision) as described previously [20].
The kit can detect 10 pmol5 nmol of acetylcholine. In
brief, the isolated smooth muscle tissue was homogenized
in Choline Assay Buffer and centrifuged at 6009g for
30 min to remove debris. The supernatant was tested by
mixing enough reagents in each well. A total of 50 ll
reaction mixture contains 44 ll Choline Assay Buffer, 2 ll
choline probe, 2 ll acetylcholinesterase and 2 ll enzyme
mixture. All the samples were incubated at room temperature for 30 min in dark room and measured OD at 570 nm
for the colorimetric assay in a micro-plate reader (Wallac
1420 vector2, PerkinElmer, Inc., San Jose, CA, USA). We
subtracted the background value (the 0 choline control)
from all standard and sample readings.
The bladder ATP content was measured using a luciferin-luciferase assay kit (Roche, Penzberg, Germany)
according to the manufacturers instructions and methods
published previously [21]. The method was used to measure the ATP dependency of the light-emitting luciferasecatalyzed oxidation of luciferin. The isolated smooth
muscle tissue was homogenized using liquid nitrogen and
diluted in a buffer containing 100 mM Tris and 4 mM
EDTA (pH 7.75) and mixed immediately with equal
amounts of the luciferase reagent. The light emitted from
the luciferase was measured using a Chemiluminescence
Analyzing System (CLD-110, Tohoku Electronic In. Co.,
Sendai, Japan), and the values were calibrated against a
standard ATP curve. Furthermore, we used BioVisions
ATP Colorimetric/Fluorometric Assay Kit (Catalog
#K354-100) to further determine ATP content in the
muscle layer of the urinary bladder. In brief, for the colorimetric assay, the ATP standard was prepared to obtain 0,
2, 4, 6, 8, 10 nmol/well of ATP standard. The bladder
tissue was homogenized using perchloric acid (BioVision,
Cat. # K808-200) and centrifuge ice cold at 15,0009g for
2 min to collect supernatant. The supernatant was mixed
with 44 ll ATP Assay Buffer, 2 ll ATP probe, 2 ll ATP
converter and 2 ll developer mix. All the samples were
incubated at room temperature for 30 min in dark room and
measured OD at 570 nm for the colorimetric assay in a
micro-plate reader (Wallac 1420 vector2). We subtracted
background value (the 0 ATP control) from all standard
and sample readings.
Electron microscopic analysis
Bladder tissues from 4 groups of rats were performed
electron microscopic observation. The tissue for electron
microscopy was fixed in half-strength Karnovskys solution (1 % paraformaldehyde and 1.25 % glutaraldehyde in
0.1 M Na cacodylate buffer, pH 7.0), postfixed in osmium

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tetroxide, dehydrated in graded ethanol and embedded in

epoxy resin. These thin tissue sections were stained with
uranyl acetate and lead citrate and examined with a camera
system (Advanced Microscopy Techniques, Danvers, MA,
USA). The ultrastructure especially in mitochondria of the
bladder specimen was photographically documented as
described previously [16].
Mast cell, cytochrome C and PGP 9.5 staining
We performed toluidine blue staining for specific mast cell
labeling [22], cytochrome C immunostaining to evaluate
mitochondrial injury, and an antiubiquitin carboxyl terminal hydrolase antibody, PGP 9.5, to evaluate the nerve
distribution in urinary bladder of four groups of rats (n = 3
each) [12]. In brief, the urinary bladder was perfused with
Tris-buffered saline and fixed with a cold mixture of 2 %
paraformaldehyde, lysine and sodium periodate for two
hours. Tissues were OCT embedded, and frozen sections
were obtained. After drying for two hours, sections were
rehydrated and washed with Tris-buffered saline. We
incubated rehydrated sections with toluidine blue solution
(Polysciences, No. 1234, 1 g/100 ml of 70 % ethanol
stock) diluted 1:10 with 1 g/ml aqueous NaCl for 2 min
followed by 3 rinses with deionized water. Specific mast
cell stain was demonstrated in blue color under microscopic observation.
The frozen sections of OCT-embedded tissues were
incubated overnight at 4 C with polyclonal cytochrome C
antibody (Santa Cruz Biotechnology, Inc.) and antiubiquitin carboxyl terminal hydrolase antibody (CEDARLANE
Laboratories Limited, Burlington, ON, Canada, 1:1,000).
The reaction was visualized by the avidinbiotinperoxidase complex method. These stains were taken by a
microscopy (Leica CM1900). The value of mast cells and
PGP 9.5 stain/total section area was counted by Adobe
Photoshop 7.0.1 image software analysis.

Eur J Nutr (2013) 52:347359

using an image analyzing system (Alpha Innotech, San

Leandro, CA).
Statistical analysis
Values are displayed as means standard error mean
(SEM). Data were subjected to analysis of variance, followed by Duncans multiple-range test for assessment of
the differences among groups. Students paired t test was
used to detect differences between control and drug treatment. P \ 0.05 was considered to indicate statistical
significance.

Results
Baseline parameters
The baseline physiologic and biochemical data of four
groups of male Wistar rats are indicated in Table 1. There
were no significant differences of body weights of the
fructose-fed rats and control animals in both 3- and
6-month groups. However, the level of triglycerides, lowdensity lipoprotein (LDL) and fasting glucose was significantly elevated in 3- or 6-month fructose-fed rats versus
age-matched control rats. We further confirmed that a
marked lipid accumulation by oil red O staining in the
livers of 3-month (Fig. 1b) and 6-month fructose-fed rats
(Fig. 1d). The mean changes of hepatic oil red stain in
these rats were demonstrated in Fig. 1e. The mean arterial
pressure showed a tendency to be increased but was not
significantly increased in the 3-month fructose-fed rats.
However, mean arterial blood pressure was significantly
increased after 6-month fructose feeding. The significantly
elevated levels in plasma triglyceride, LDL, fasting glucose
and mean arterial blood pressure were found in our fructose-fed male rats indicating the successful induction of
metabolic syndrome.

Western blot
Altered cystometric data after fructose feeding
We evaluated the expression of M2- and M3-muscarinic
cholinergic receptors, P2X1, P2X2, and P2X3 receptors of
bladder smooth muscle layer tissues by a Western blotting
technique as described previously [23]. In brief, the muscle
samples were homogenized with a prechilled mortar and
pestle in extraction buffer and kept at 4 C for 30 min. The
antibodies against M2 (AB5166, Chemicon International,
CA), M3 (Alomone Labs, Israel), P2X1 (Santa Cruz Biotechnology, Germany), P2X2 (Neuromics, Northfield, MN),
P2X3 receptor (Santa Cruz Biotechnology, Germany) and
b-actin (Sigma, Saint Louis, MO) were indicated. The
density of the band with the appropriate molecular mass
was determined semi-quantitatively by densitometry

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In the present study, we selected 3- or 6-month fructose-fed

male rats (n = 8 each) with LUTS to examine its possibly
pathophysiologic mechanisms in comparison with respective age-matched control rats. We have demonstrated the
original diagrams in Fig. 2 and showed that several cytometric parameters are altered after long-term fructose
feeding. As listed in Table 1, the LUTS characterized by
decreased ICI and pIVP together with increased BP and TP
are present in 3- or 6-month groups of fructose-fed rats, but
not in the age-matched control groups.
More specifically, we found that the contractile components of phase 1 and duration in phase 2 in fast chart

Eur J Nutr (2013) 52:347359

351

Table 1 General characteristics in the control and fructose-fed animals

Study groups

#3 months
Chow

n
Body weight (gm)
Triglycerides (mg/dl)
Cholesterol (mg/dl)
HDL (mg/dl)

6
443 10.0
57 9

#6 months
FFD

Chow

418 14

0.2

489 18

480 20

114 6

0.001

44 5

94 3

FFD

8
0.8
0.001

46.8 6.8

57.7 5.1

0.3

48 4

39 5

0.54

22 1.9

26.8 1.4

0.086

17 0.9

18 1

0.83

LDL (mg/dl)

3.7 0.5

7.2 0.4

0.05

4.8 0.6

7.5 0.3

0.05

Fasting glucose (mM)

Blood pressure (mmHg)

6.3 0.5
105 10

7.4 0.5
115 10

0.043
0.148

6.2 0.4
102 10

7.3 0.4
130 10

0.048
0.035
0.02

ICI (s)

190 79

72 17

0.02

182 65

74 21

TP (cmH2O)

16.3 0.7

22.8 1.2

0.001

18.4 2.5

29.3 0.2

0.002

BP (cmH2O)

14.2 1.6

20.6 1.9

0.001

17.0 2.4

30.0 0.9

0.001

IVP (cmH2O)

39.1 4.4

29.3 0.1

0.031

38.9 3.6

30.5 3.3

0.026

Value are expressed as mean SEM. FFD fructose feeding, HDL high-density lipoprotein, LDL low-density lipoprotein, ICI intercontraction
interval, TP threshold pressure, BP baseline bladder pressure, IVP peak intravesical pressure

Fig. 1 Long-term fructose

feeding increases hepatic lipid
accumulation. ad Hepatic lipid
accumulation by oil red stain.
The hepatic fat area stained by
oil red O was increased in the
3-month (3 M) (b) or 6-month
(6 M) (d) fructose feeding when
compared to 3 M (a) or 6 M
(c) normal controls

speed are changed after 3- (Fig. 2d) or 6-month (Fig. 2h)

fructose-feeding versus respective control groups (Fig. 2c,
g). Depressed phase 1 contractile amplitude and phase 2
contractile duration in the urinary bladder are markedly
indicated in the 3- or 6-month fructose-feeding rats. The
phase 2 contraction almost disappeared in the voiding
contraction after 6-month fructose feeding (Fig. 2h).
The original recordings of pelvic afferent and efferent
nerve activities, extraurethral sphincter-electromyogram
activity and IVP in the 3-month control rat, 3- and 6-month

fructose-feeding rats were displayed in Fig. 3a. In the control rats, saline infusion gradually enhanced pelvic afferent
nerve activity and extraurethral sphincter-electromyogram
activity and increased IVP, but did not activate pelvic
efferent nerve activity during the filling. At phase 1 for
voiding contraction, pelvic afferent nerve activity increased
sharply in parallel with the rising IVP and enhanced tonic
pelvic efferent nerve activity and extraurethral sphincterelectromyogram activity. During phase 2 contractions
of voiding function, pelvic efferent nerve activity and

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Eur J Nutr (2013) 52:347359

Fig. 2 Alterations in
micturition parameters in 3- and
6-month fructose-fed groups.
Cystometric parameters are
indicated in 3-month control
group (a, c), 3-month fructosefed group (b, d), 6-month
control group (E&G) and
6-month fructose-fed group
(f, h). c, d, g, h are the graphs
(marked with asterisk from
a, b, e, f) with fast chart speed.
Phase 1 and 2 contractile
responses are well reserved in
3-month (c) and 6-month
control group (g), whereas
phase 1 and 2 contractile
responses are depressed in
3-month (d) and 6-month
fructose feeding (h).
BP baseline bladder pressure,
TP threshold pressure,
ICI intercontraction interval,
pIVP peak value of intravesical
pressure

extraurethral sphincter-electromyogram activity fired in

burst discharges and IVP displayed high frequency oscillations. After phase 2 contractions, the increased pelvic
afferent and efferent nerve activity, extraurethral sphincterelectromyogram activity and IVP were reduced and returned
to their baseline levels. After 3-month fructose feeding,
there was a decrease in IVP level in phase 1 contraction, a
decrease in voiding duration in phase 2 contraction and a
longer enhancement in pelvic afferent nerve activity. After

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6-month fructose feeding, there was a decreased IVP, a

shortened phase 2 contractile duration, and an alteration in
pelvic afferent nerve activity, pelvic efferent nerve activity
and extraurethral sphincter-electromyogram activity. The
recordings of pelvic afferent nerve activity, pelvic efferent
nerve activity, extraurethral sphincter-electromyogram
activity and IVP showed that 3- or 6-month fructose feeding
markedly altered neural and myogenic activity during a
voiding contraction when compared to control group.

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353

Fig. 3 a An integrated graph of simultaneous recordings of PANA,

PENA, EUS-EMG and IVP during a continuous cystometrogram
(0.10 ml/min infusion) in three groups of rats is demonstrated.
Abnormal pattern of PANA, PENA, EUS and IVP (indicated by a) is
found in fructose-fed rats when compared to the control rat. The
urinary bladder ATP content measured by luciferin-luciferase assay
(b) and by ATP colorimetric assay (c) indicated that the ATP content

in smooth muscle of urinary bladder is significantly decreased after

3-month (3 M) or 6-month (6 M) fructose feeding when compared to
respective normal control group. The acetylcholine content in smooth
muscle of urinary bladder is also significantly depressed after 3-month
(3 M) or 6-month (6 M) fructose feeding when compared to normal
controls (d). *P \ 0.05 when compared to respective control group

We further evaluated the content of ATP and acetylcholine, two major neurotransmitters contributing to bladder contractions, in the normal and fructose-feeding rats.
The level of ATP in 3- and 6-month normal controls was
8.42 0.27 and 8.09 0.28 lg/mg tissue by luciferinluciferase assay (Fig. 3b) and 3.27 0.87 and 3.17
0.78 nmol/mg bladder tissue by ATP colorimetric assay
(Fig. 3c), respectively. The ATP level of 3- and 6-month
fructose feeding was 6.68 0.29 and 6.12 0.42 lg/mg
tissue by luciferin-luciferase assay and 1.58 0.0.31 and
1.29 0.24 nmol/mg bladder tissue by ATP colorimetric
assay. The level of acetylcholine of the urinary bladder
in 3- and 6-month normal controls was 76 3 and 74 9
nM/mg tissue (Fig. 3d) and that was significantly decreased
to 34 3 and 35 6 nM/mg tissue after 3- and 6-month
fructose feeding.

toluidine blue stain was found in the 3- and 6-month

fructose-fed rats versus 3- and 6-month normal-diet rats.

Increased mast cells in the bladder after fructose

feeding

Ultrastructure analysis by electron micrography

We found that the mitochondrial structure is markedly
altered with the disrupted and swollen characteristics
(Fig. 4j, l) in the smooth muscles of fructose feeding
versus mitochondria in controls (Fig. 4i, k). Cytochrome
C, a marker for mitochondrial injury, is markedly stained
in the 3-month (Fig. 4n) and 6-month (Fig. 4p) fructosefed bladder smooth muscles when compared to 3- and
6-month controls (Fig. 4m, o). The semi-quantitative
analysis of cytosolic cytochrome C by Western blot
showed that the cytosolic cytochrome C expressions were
significantly enhanced in the 3- and 6-month fructose-fed
bladders than those in the 3- and 6-month control bladders
(Fig. 5c).
Reduced nerve density in smooth muscle layer

An increased mast cell number located in the interstitium

or around the blood vessels was found in the urinary
bladder after 3-month (Fig. 4b, f) or 6-month (Fig. 4d, h)
fructose-fed rats, but not in 3-month (Fig. 4a, e) or 6-month
control rats (Fig. 4c, g) by toluidine blue and H&E stain,
respectively. The quantitative data of the mast cells were
shown in the Fig. 5a. A significant increase in mast cells by

To address whether alterations in nerve density of bladder

smooth muscles after fructose feeding, we used antibody
PGP 9.5 to stain nerve fibers. Noted that the stained fibers
were found in the muscles and vessels of the 3-month
(Fig. 4q) or 6-month control urinary bladder (Fig. 4s).
A decreased density of PGP 9.5 stained fibers was seen in

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Fig. 4 Effects of long-term fructose feeding on morphological

alterations and bladder nerve density in the urinary bladder.
ad Specific mast cell staining (indicated by arrows) by toluidine
blue dye. eh Morphologic changes and increased mast cells in the
fructose-feeding bladder (marked with arrows) by H&E stain.
il Disrupted, swollen and bloated mitochondria (marked with
arrows) found in the smooth muscles of 3-month (j) and 6-month

(l) fructose-treated bladder by electron micrography. mp Marked

cytochrome C stain (brown color) in the muscle of 3- (n) and 6-month
(p) fructose-fed groups when compared to 3- (m) and 6-month
(o) controls. qt A decrease in nerve innervations in the smooth muscles of 3-month (r) and 6-month (t) fructose-treated urinary bladder
by PGP 9.5 stain (brown color) when compared to 3- (q) and 6-month
(s) controls

the smooth muscle layer of urinary bladder after 3-month

(Fig. 4r) or 6-month fructose feeding (Fig. 4t). The quantitative data of PGP 9.5 density was displayed in the

Fig. 5b. A significantly decreased PGP 9.5 stain was noted

in 3- and 6-month fructose-fed bladders than that in 3- and
6-month controls.

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Fig. 5 Quantitative data of mast cell numbers, PGP 9.5 denisty and
cytosolic cytochrome C (C cyt c) stain by Western blot in urinary
bladders of the normal controls and fructose-feeding rats. a Statistic
data of toluidine blue positive cells in the urinary bladders.
b Quantitative data of PGP 9.5 density in the smooth muscles of
urinary bladders. c Original graph and semi-quantitative data of the C
cyt c from the normal control and fructose-feeding rats. *P \ 0.05
when compared to respective control group

Upregulation of purinergic and muscarinic cholinergic

receptors after fructose feeding
Figure 6 demonstrates representative Western blotting
results and statistic comparisons for M2 and M3 muscarinic
cholinergic receptors protein expression in the smooth

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Fig. 6 Western blot of M2 (a) and M3 receptor (b) protein expression

in the smooth muscle of urinary bladders from control and fructosefed rats. After 3 or 6 months of fructose feeding, the M2 and M3
receptor protein expression in the urinary bladder is significantly
enhanced when compared to respective control group. *P \ 0.05
when compared to respective control group

muscles between controls and fructose-fed groups. Significant upregulation of M2 (Fig. 6a) and M3 muscarinic
cholinergic receptors (Fig. 6b) was implicated in the 3- and
6-month fructose-fed groups in comparison with respective
control groups. As shown in Fig. 7, significant upregulation
in P2X1 (Fig. 7a), P2X2 (Fig. 7b) and P2X3 (Fig. 7c)

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Fig. 7 Western blots of P2X1

(a), P2X2 (b) and P2X3 receptor
(c) in the smooth muscles of
urinary bladders of control and
fructose-fed animals. After 3 or
6 months of fructose feeding,
the P2X1, P2X2 and P2X3
receptor protein expression in
the urinary bladder is
significantly enhanced when
compared to respective control
group. *P \ 0.05 when
compared to respective control
group

receptor protein expression is also noted in the fructose-fed

rats when compared to respective control group.

Discussion
The consumption of fructose has been reported to
increase the metabolic syndrome, because fructose triggers hypertriacylglycerolemia, hyperglycemia and hypertension [6, 11, 16, 24], which are consistently found in
our animals. The body weight gain was not different after
3- or 6-month fructose feeding. These results are in
accordance with previous reports, which demonstrated
that fructose intake did not further increase the body
weight [2527].
A clinical finding indicated the storage/voiding symptoms including frequency, urgency, weak stream, intermittency, and incomplete emptying occurred in the LUTs
patients with metabolic syndrome [7]. Martin et al. [7]

123

indicated that the functional decline of the lower urinary

tract is related with the factors including abdominal fat
mass, plasma glucose, HDL cholesterol and energy intake.
These factors positively associated with storage LUTS
were increased abdominal fat mass, plasma glucose and
low HDL cholesterol. Overconsumption of sugar-sweetened beverages, in particular carbonated soft drinks, promotes the development of overweight and obesity and is
associated with metabolic disturbances, including intrahepatic fat accumulation and metabolic syndrome [28].
Hyperlipidemia induced by a high-fat diet is associated
with increased urinary frequency, and decreased bladder
blood vessel and nerve density in rats [29]. The alterations
in the bladder smooth muscle, neuronal degeneration and
urothelial dysfunction have also been observed in a type II
diabetes mellitus rat model induced by a high-fat diet and
low-dose streptozotocin [30]. Our serial reports have found
that fructose-induced metabolic syndrome induces LUTS
in our animal models. These reports and our data evidence

Eur J Nutr (2013) 52:347359

that metabolic syndrome characterized with hyperlipidemia

and hyperglycemia is associated with LUTS.
Our results were the first to determine the long-term
fructose effects on the neural elements contributing to
voiding function by simultaneously recording cystometrogram, pelvic afferent and efferent nerve activity and
extraurethral sphincter-electromyogram activity in the
urethane-anesthetized rats. According to our data and other
report [13, 31], voiding function is activated by distensioninduced pelvic afferent nerve activity from the bladder
receptors, triggered the neurons located in the sacral spinal
cord and elicited the early central reflex of tonic firing of
pelvic efferent nerve activity and extraurethral sphincterelectromyogram activity in phase 1. Tonic pelvic efferent
nerve activity may release ATP and acetylcholine and leads
to contraction of the detrusor muscle, whereas tonic
excitation of the extraurethral sphincter-electromyogram
activity contributes to the tight closure of the sphincter, thus
increasing the IVP. Fluid emission was confined almost
completely in phase 2, when a series of high frequent
oscillations accompanied the acetylcholine-dependently
bursting pelvic efferent nerve activity and extraurethral
sphincter-electromyogram activity [13]. Alterations in the
firing types of pelvic afferent nerve activity, pelvic efferent
nerve activity and extraurethral sphincter-electromyogram
activity have been demonstrated in bladder dysfunction like
substance P-induced hyperactive bladder [12] and bladder
outlet obstruction [13]. In the fructose-induced LUTS, we
recognized the suppression in phase 1 contractile amplitude,
phase 2 voiding duration and the alteration in pelvic afferent
nerve activity, pelvic efferent nerve activity and extraurethral sphincter-electromyogram activity. ATP is responsible
for mediating phase 1 detrusor contraction, whereas acetylcholine is required for regulating phase 2 detrusor contraction [13]. According to our findings, the decreased ATP
and acetylcholine content in the smooth muscle of the urinary bladder are found after long-term fructose feeding. The
reduction in ATP and acetylcholine may participate in the
alteration of pelvic nerve activity and result in metabolic
syndrome-induced LUTS.
How fructose feeding reduces ATP and acetylcholine
content in the urinary bladder? Fructose produces deleterious metabolic effects in animal models. There are some
possible mechanisms for the reduction in these neurotransmitters. In the urinary bladder, a decrease in ATP
content could be due to a metabolic effect or resulted from
a consequence of metabolic syndrome. ATP can be
depleted either in primary hepatocytes or in the liver in
vivo by fructose treatment directly because of its metabolic
effect [32]. Another reason is that the metabolic syndrome
with hyperlipidemia and hyperglycemia impair mitochondrial structure and function for incapability to producing
ATP in the long-term fructose-fed urinary bladder smooth

357

muscles. Nevertheless, it requires further experiments to

explore the mechanisms for fructose feeding on reducing
bladder ATP and acetylcholine concentration.
ATP contributes to excitatory neurotransmission in the
urinary bladder through P2X receptors in detrusor smooth
muscle [33, 34]. P2X2 and P2X3 receptors play unique
and tissue-specific roles in micturition reflex pathways.
Upregulation of P2X2 receptors in detrusor smooth muscle
from patients with idiopathic detrusor instability has been
indicated [33]. Our data and other reports have found the
changes in postsynaptic P2X1 and P2X3 receptors [16, 24]
expression in the urinary bladders of female rats after longterm fructose feeding. P2X3 receptor knockout mice exhibit
bladder hyporeflexia on cystometry with decreased voiding
frequency and increased bladder capacity and voided volume but normal bladder pressures [34]. On the other hand,
acetylcholine, another important neurotransmitter, via M2
and M3 receptors regulates micturition. Some reports [11,
16] indicated fructose feeding increased postsynaptic M2
and M3 muscarinic cholinergic receptors in the smooth
muscle layer of the female rats. In the present study, we
further found upregulation in P2X1, P2X2, P2X3 and M2 and
M3 muscarinic cholinergic receptors in the male rats with
long-term fructose feeding. Longer fructose feeding has a
tendency to further enhance these postsynaptic receptors
expression. In vitro study shows that 6-month fructose
treatment significantly reduced the contractile amplitude to
filed stimulation, KCl and carbachol, but increased the
contractile response to high [ATP] ([5910-4 M) in the
isolated detrusor from Wistar female rats [16]. Alterations in
purinergic and cholinergic pathways have found in some
pathophysiological states of bladder, such as aging, diabetes
or spinal cord injury [3537]. We implicate the alterations in
purinergic and muscarinic cholinergic receptors in the urinary bladder smooth muscles possibly contribute to fructose
feedinginduced neurogenic and myogenic changes.
Mitochondria are one of major sources of reactive oxygen species (ROS) in cells, and mitochondrial dysfunction
increases the generation of ROS from the mitochondrial
respiratory chain. Hyperglycemia and hyperlipidemia
increased the expression of NADPH oxidase and ROS
release [8]. Increased oxidative stress causes intramural
bladder nerve dysfunction, nerve fiber injury, mitochondrial
injury and detrusor muscle cell damage [38]. In substance
P-induced bladder hyperactivity [12], increased superoxide
anion evokes alterations in pelvic afferent nerve activity,
pelvic efferent nerve activity and IVP responses, and the
use of superoxide dismutase attenuates these alterations
indicating oxidative stress contributing to bladder hyperactivity. Mitochondrial oxidative stress resulting from
hyperlipidemia or hyperglycemia is additive and directly
injures dorsal root ganglion neurons via interaction with the
receptor lectin-like oxLDL receptor-1 and NAD(P)H

123

358

oxidase activity [9]. Oxidative stress increased the susceptibility of muscle to the accumulation of mitochondrial
damage [39]. Metabolic syndrome in our model characterized with hyperglycemia, hyperlipidemia and hypertension
could increase oxidative stress and impair mitochondrial
structure [24] and function in axons and smooth muscles,
which may block axonal transport and neurotransmitter
production/release in the damaged pelvic nerves and
depress detrusor cell contractility. This injury could also
possibly result in the conspicuous degenerative in axons,
neuromuscular junction and the reduction in the synaptic
vesicles for production/release of ATP and acetylcholine.
Our data found a decrease in PGP 9.5 nerve density in
fructose-treated bladder. The alterations in pelvic nerve
activity and density may also contribute to urinary bladder
dysfunction.
On the other hand, the death of smooth muscle cells is
associated with markedly decreased mitochondrial transmembrane potential as well as upregulation of cellular
ROS and cytosolic cytochrome C release [10]. Our results
have demonstrated the abnormally structural mitochondria
and observed the increased cytosolic cytochrome C stain
and expression in the fructose-treated smooth muscles. In
myogenic injury, our previous study has shown that a
down-regulation of anti-apoptotic Bcl-2 protein and an
increased Bax/Bcl-2 ratio/caspase 3 signaling enhances an
intrinsic apoptotic pathway in the smooth muscles of
fructose-fed rats [24]. The increased apoptosis formation
promotes the loss of bladder smooth muscle cells, which
have been recognized by the decreased smoothelin
expression, a novel cytoskeletal protein specific to smooth
muscle cells, in the bladder smooth muscles of fructose-fed
rats [24]. Furthermore, the decreased in pIVP of the urinary
bladder smooth muscles in phase 1 and loss of phase 2
found in our fructose-fed rats may indicate fructoseinduced injury in the smooth muscles.
Increased mast cell number and histamine release in the
kidney impaired neural afferent signaling after diabetes
[40]. After fructose feeding, we also found increased mast
cells in the urinary bladder. We suggest the increased mast
cells possibly via the action of increased histamine release,
inflammatory cytokines or oxidative stress to impair neuronal and muscular function in the LUTS. The rat bladder
contains approximately 16,000 axons of which at least half
are sensory and approximately 3/4 of the mitochondria of a
nerve are primarily located in axons [41]. Our data found
significantly reduced nerve density in smooth muscle layer
after 3 and 6 months of fructose feeding. Decreased sensory
and sympathetic innervation may affect filling capacity
resulting in micturition frequency, whereas reduced parasympathetic innervation could decrease micturition pressure
for incomplete emptying. This will be further determined in
future.

123

Eur J Nutr (2013) 52:347359

In conclusion, long-term fructose feeding induced LUTS

associated with the alterations in firing type of pelvic nerve
and external urethral sphincter activity. Increased mast cell
numbers, impaired mitochondrial integrity, reduced nerve
distribution, ATP and acetylcholine content and upregulation of postsynaptic purinergic and muscarinic cholinergic
receptors expression may play a role in long-term fructose
feedinginduced bladder dysfunction.
Acknowledgments This work was supported by the National Science Council of the Republic of China (NSC99-2314-B418-002-MY3
and NSC99-2628-B-002-058-MY3) and research fund from FarEastern Memorial Hospital (FEMH-99-D-006).
Conflict of interest

None.

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