Vol.2, No.9, 1072-1077 (2010) Health
doi:10.4236/health.2010.29157
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Reduced bile duct contractile function in rats with
chronic hyperglycemia
Chi-Ming Liu1, Hui-Chen Su2, Yen-Ting Wang1, Tao-Hsin Tung1, Pesus Chou3, Yiing-Jeng
Chou3, Jorn-Hon Liu1, Jan-Kan Chen4*
1Cheng-Hsin General Hospital, Taipei, Taiwan, China
2Chi-Mei Medical Center, Tainan, Taiwan, China
3Institute of Public Health, National Yang Ming University, Taipei, Taiwan, China
4Department of Physiology, College of Medicine, Chang Gung University, Taipei, Taiwan, China;
*Corresponding Author: jkc508 @ mail. cgu.edu.tw
Received 27 November 2009; revised 11 January 2010; accepted 3 March 2010.
ABSTRACT
The incidence of gallstone is higher in patients
with diabetes mellitus than in general popula-
tion. It is generally attributed to hypomotility
and lowered emptying function of the gallblad-
der. In this study, we investigate if chronic hy-
perglycemia is correlated with reduced contrac-
tile function of the bile ducts in rat. Hypergly-
cemic rats were induced by streptozotocin-nic-
otinamide treatment. Hyperglycemic rats were
sacrificed eight months after induction and bile
ducts were removed for the subsequent studies.
The bile duct contractility of the normal rats is
consistently higher than that of the hypergly-
cemic rats. The contractities were measured to
be 5.5 ± 0.2 mg vs. 4.2 ± 0.1 mg without CCK
stimulation, and 5.5 ± 0.3 mg vs. 7.9 ± 0.4 mg
with CCK stimulation, respectively for hypergly-
cemic and normal rats. There was no significant
difference in plasma CCK concentration in hy-
perglycemic rats and normal rats. The expres-
sion of CCK-A receptor protein in the bile duct
tissue was decreased in hyperglycemic rats
compared with that of the normal rats, and it
may, at least in part, responsible for a reduced
contractility. A reduced bile duct motility may
cause bile retention, and may be one of the
factors predispose to gallstone formation in
type 2 diabetes patients, which is characterized
with chronic hyperglycemia.
Keywords: Hyperglycemia; Bile Duct; Contractile
1. INTRODUCTION
Cholelithiasis is one of the most prevalent gastroentero-
logic diseases in humans. It is a complex metabolic dis-
orders, and its exact pathogenic mechanisms have not
been fully elucidated. Gallstones represent a serious
burden for the health care systems: over 10% of Euro-
peans and Americans carry gallbladder stones [1], and
the prevalence of gallstone disease seems to be rising as
a result of longer life expectancy [2]. Many gallstones
are silent, but symptoms and severe complications en-
sure in around 25% of the cases, necessitating surgical
removal of the gallbladder [3]. Mortality rates following
cholecystectomy range from less than 0.1% in clinical
studies [4] to 0.8% (as documented for all cholecystec-
tomies performed in Germany in 2002) [5]. In the US
about 3,000 deaths (0.12% of all deaths) per year are
attributed to complications of cholelithiasis and gall-
bladder disease [6]. Nonsurgical approaches, including
gallstone dissolution by ursodeoxycholic acid and ex-
tracorporeal shockwave lithotripsy, have increasingly
lost their impact on therapy and are performed only for
uncomplicated symptomatic cholecystolithiasis in a very
small number of selected patients.
Bile formation enables the removal of excess choles-
terol, either directly or after catabolism to bile salts, and
it is a key function of the liver. Bile is an aqueous solu-
tion of lipids, in which bile salts (67% of solutes by
weight), phospholipids (22%) and cholesterol (4%) rep-
resenting the three main lipid species [7]. More than
80% of gallstones consist mainly of cholesterol and are
formed within the gallbladder [8]. Three major mecha-
nisms contribute to the formation of cholesterol gall-
bladder stones: cholesterol supersaturation of bile, gall-
bladder hypomotility and destabilization of bile by ki-
netic protein factors. Cholesterol-supersatu-rated bile
contains more cholesterol than can be solubilized by
mixed micelles (cholesterol saturation index > 1). It
contains multilamellar vesicles (liquid crystals), whose
*Chi-Ming Liu and Hui-Chen Su contributed equally in this study.

C.-M. Liu et al. / HEALTH 2 (2010) 1072-1077
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
1073
fusion and aggregation precede the formation of solid
cholesterol crystals. As illustrated in the classic triangu-
lar phase diagram, solid crystals occur in bile at high
relative bile salt and low phospholipids concentrations
and at cholesterol: phospholipids ratios > 1 [7]. An ex-
cess of biliary cholesterol in relation to bile salts and
phospholipids can result from hypersecretion of choles-
terol, or from hyposecretion of bile salts or phospholip-
ids. Cholesterol hypersecretion is the most common
cause of supersaturation [8]. It might be caused by in-
creased hepatic uptake or synthesis of cholesterol, de-
creased hepatic synthesis of bile salts, or decreased he-
patic synthesis of cholesteryl esters for incorporation in
VLDL. Accordingly, any enzyme, transporter or regula-
tor involved in hepatic cholesterol metabolism could
potentially affect the formation of cholesterol gallstones
[9]. In humans, most gallstone cholesterol is of dietary
origin, consistent with the observation that hepatic bio-
synthesis contributes less than 20% of the biliary cho-
lesterol [2]. The hepatic uptake of cholesterol is medi-
ated by the scavenger receptor B1 for HDL, which con-
tributes most of the biliary cholersterol under physiol-
ogic conditions. The inverse correlation between serum
HDL levels and gallstones suggests that cholesterol
cholelithiasis is associated with an induced reverse cho-
lesterol transport and hepatic catabolism of HDL [2].
The rate-limiting enzymes of hepatic cholesterol and bile
salt synthesis are 3-hydroxy-3-methylglutary-coenzyme
A reductase and cholesterol 7α-hydroxylase, respectively.
These enzymes are regulated by the sterol-regulatory-
lement-binding protein (SRBP) and nuclear receptor
signaling pathways [10,11]. Stasis of bile in the gall-
bladder favours stone formation, as indicated by stone
formation during pregnancy, rapid weight loss or total
parenteral nutrition. Postprandial gallbladder volumes
are increased and gallbladder emptying in response to
cholecystokinin (CCK) is impaired in patients with gall-
stones [8], probably as a result of absorption of choles-
terol from supersaturated bile by gallbladder wall. Ex-
cess cholesterol in smooth-muscle cells stiffens sar-
colemmal membranes and decouples the G-protein-me-
diated signal transduction of the CCK, thereby paralyz-
ing gallbladder contractile function [12].
Among the diabetes patients in China, about 10% of
them also bear gallstones. The impaired emptying func-
tion owing to hypomotility of the gallbladder is consid-
ered an important factor for the development of chole-
lithiasis [13-17]. Because of hypomotility and lowered
emptying function, the incidence of gallstone is higher in
patients with diabetes mellitus than in general population,
however, its underlying mechanism has not been well
understood.
Gallbladder mortility is regulated by cholinergic and
gastrointestinal hormone [18,19]. Postprandial gallblad-
der emptying is triggered mainly by plasma CCK from
small intestine. CCK interacts with CCK receptor-1
(CCK-R) in gallbladder smooth muscle cells, which in
turn elicits the contraction of gallbladder by the activa-
tion of post-membrane signaling passway [20]. We hy-
pothesize that abnormal gallbladder contraction in re-
sponse to CCK, may play an important role in the deve-
lopment of cholesterol gallstone in hyperglycemic pa-
tient. The purpose of this study was to examine if there
are differences in gallbladder motor function, plasma
CCK concentration, and the CCK-R activity in response
to CCK in hyperglycemic and normal rats.
2. MATERIALS AND MATHODS
2.1. Animals
Male SD rats, age 8-10 week, obtained from the Na-
tional Laboratory Animal Center, Taiwan, were used for
the study. Streptozotosine (STZ), which selectively de-
stroys the pancreatic β-cells that secrete insulin, was
used to induce insulin dependent DM, and nicotinamide
was used to attenuate STZ effect to induced insulin-de-
ficient diabetic rats [21]. STZ-nicotinamide DM rats
were induced in overnight fasted animals by a single
intravenous injection of STZ (60 mg/kg body weight),
and nicotinamide (120 mg/kg body weight) (Sigma
Chemical Co., St Louis, MO) was administered intrap-
eritoneally 15 min after STZ. Combined administration
of STZ and nicotinamide leads to the development of a
diabetic syndrome, which is characterized by moderate
and stable hyperglycemia and reduced pancreatic insulin
stores [22].
Hyperglycemia was confirmed by elevated plasma
glucose levels, measured on days 3 and 7 after drug in-
jection. STZ-nicotinamide treated rats exhibited a fasting
plasma glucose concentration of 13.4 ± 0.8 mmol/L and
a plasma insulin level of 95.0 ± 0.2 pmol/L (n = 24). In
contract, the fasting plasma glucose and insulin levels of
the normal rats were 4.8 ± 0.05 mmol/L and 168.5 ± 4.8
pmol/L, respectively (n = 24). All studies were carried
out with animals two weeks after the induction of diabe-
tes. Blood samples were taken from overnight fasted rats
at 0, 2, 4, 6 and 8 months after the confirmation of hy-
perglycemia.
2.2. Bile Duct Contractile Response
Bile ducts were isolated from hyperglycemic rats and
normal rats, respectively, and placed in Kreb’s solution
containing (in mmol/L) 118.4 NaCl, 25 NaHCO3, 11.66
glucose, 4.75 KCl, 1.18 MgSO4·7H2O, 2.5 CaCl2·2H2O,
1.19 KH2PO4, 0.02 EDTA. The solution was maintained

C.-M. Liu et al. / HEALTH 2 (2010) 1072-1077
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
1074
at pH 7.4 and continuously bubbled with 95%O2-5%CO2.
Bile ducts were carefully mounted on the isometric force
transducer in the organ chamber (95%O2-5%CO2, at
37˚C), and were equilibrated for 90 minutes in an organ
bath with a resting tension of 1.8 mg. CCK (from 10-9-
10-5 M) was used to induce bile duct contraction.
2.3. Determination of Plasma CCK
Concentration
Animals were fasted overnight and anesthetized by pen-
tobarbital (30 mg kg-1 body weight, i.p.). Blood samples
(0.1 mL) were collected from femoral vein using a
chilled syringe that contained 10 IU heparin. Plasma
CCK levels were measured by enzyme immunoassay of
50 μL aliquots of plasma with a CCK octapeptiode rat
ELISA kit (Harbor Boulevard, Belmont, California). The
immunoplate in this kit is pre-coated with secondary
antibody and the nonspecific binding sites are blocked.
The secondary antibody binds to the Fc fragment of the
primary antibody (peptide antibody) whose Fab frag-
ment will be competitively bound by both biotinylated
peptide and peptide standard or targeted peptide in sam-
ple. The biotinylated peptide is able to interact with
streptavidin-horseradish peroxidase (SA-HRP) which
catalyzes the conversion of 3,3’,5,5’-tetramethylbenzi-
dine (TMB) to produce a blue colored solution. The re-
action was stopped by adding acid and the reaction solu-
tion turned yellow and was read spectrophotometrically.
2.4. Determination of Plasma Glucose
Animals were fasted overnight and anesthetized by pen-
tobarbital (30 mg kg-1 body weight, i.p.). Blood samples
(0.1 mL) were collected from femoral vein using a
chilled syringe that contained 10 IU heparin. The sam-
ples were centrifuged at 13,000 rpm for 3 min, and ali-
quot (15 μl) of plasma was added to 1.5 ml of a Glucose
Kit Reagent (Biosystems S.A., Barcelona, Spain) and
incubated at 37˚C in a water bath (Yamato-BT-25, Tokyo,
Japan) for 10 min. Plasma glucose was determined by a
glucose analyzer (Quik-Lab, Ames, Miles Inc., Elkhart,
IN).
2.5. Determination of Plasma Insulin
Plasma insulin levels were measured by enzyme immu-
noassay of 25 μL aliquots of plasma with a Rat Insulin
ELISA kit (Mercodia, Uppsala, Sweden). During incu-
bation, insulin in the sample reacted with peroxidase-
conjugated anti-insulin antibodies which were bound to
the plastic surface of the microtitration well. The bound
conjugate was detected by reaction with 3,3’,5,5’-tetra-
methylbenzidine. The reaction was stopped by adding
acid to give a colorimetric endpoint that was read spec-
trophotometrically.
2.6. Western Blot Analysis
Bile ducts were homogenized by mechanical homogeni-
zation using a glass/Teflon homogenizer. Protein content
was determined using the BCATM protein assay kit
(Rockford, USA). A total of 50 μg of tissue protein was
fractionated on 10% sodium dodecyl sulfate/polyacryla-
mide gel electrophoresis (SDS/PAGE). Following trans-
fer, the membrane was washed with phosphate-buffer
saline (PBS) and blocked for 1 h at room temperature
with 5% (w/v) skimmed milk in PBS. Blots were then
incubated overnight at 4˚C with the polyclonal antibody-
ies against rat bile duct CCK-A or CCK-B (both at
1:1000) (abcam Littleton, CO). β-actin was also blotted
with mouse monoclonal antibody (1:1000) against β-actin
and served as internal reference. After removing the
primary antibody, the blots were extensively washed
with PBS/Tween 20, and incubated for 1 h at room tem-
perature with the appropriate peroxidase-conjugated
secondary antibody. Following removal of the secondary
antibody, blots were washed as described and developed
by autoradiography using the ELC-Western blotting sys-
tem (Amersham Corp.). Densities of the obtained im-
munoblots at 50 KDa for CCK-B, 48 KDa for CCK-A
and 42 KDa for β-actin were quantified using a laser
densitometer.
2.7. Statistical Analysis
Data are expressed as mean ± SEM (standard error of the
mean). Repeated measures of analysis of variance
(ANOVA) were used to analyze the changes in plasma
glucose and other parameters. The Dunnett range of post
hoc comparisons was used to determine the source of
significant differences where appropriate. A p value of
0.05 or less was considered as significant.
3. RESULTS
3.1. Measurement of the Plasma Glucose
and Insulin Levels
Plasma glucose and insulin levels were measured from
blood samples taken from overnight fasted rats. The
blood glucose concentrations measured at 0, 2, 4, 6 and
8 months after confirmation of the hyperglycemia were
13.4 ± 0.8, 14.4 ± 0.6, 15.6 ± 0.2, 16.2 ± 0.1, 16.6 ± 0.2
mmol/L, respectively (Figure 1). The plasma insulin
concentrations measured at the same time intervals after
confirmation of hyperglycemia were 95.0 ± 0.2, 99.5 ±
0.4, 115.2 ± 0.3, 125.6 ± 0.6 and 128.4 ± 0.8 pmol/L,
respectively (Figure 1). During the same period of time,
the plasma glucose and insulin levels of the normal rats
were 4.8 ± 0.5 mmol/L and 168.5 ± 4.8 pmol/L, respec-
tively (n = 24).

C.-M. Liu et al. / HEALTH 2 (2010) 1072-1077
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1075
16.38
13.17
14.03
15.40 15.90
115.19
128.31
95.33
99.39
125.29
0
2
4
6
8
10
12
14
16
18
02 month4 month6 month8 month
plasma glucose (mmol/L
)
80
100
120
140
plasma insulin (pmol/L
)
plasma insulin (pmol/L)
p-value for trend = 0.003
p-value for trend = 0.005
p-value for trend = 0.005
Figure 1. Plasma glucose concentrations and plasma insulin
concentrations in STZ-nicotinamide induced hyperglycemic
rats. Data are presented as Means  Std.
3.2. Measurement of the Plasma CCK
Levels
No significant difference in plasma CCK levels between
normal and hyperglyvemic rats were observed. In the fed
normal and fed hyperglycemic rats, the plasma CCK lev-
els were ranged from 0.70 ± 0.05 to 0.79 ± 0.04 ng/ml,
and 0.74 ± 0.06 to 0.78 ± 0.04 ng/ml, respectively (Fig-
ure 2(a)). In overnight fasted normal and hyperglycemic
rats, the plasma CCK levels were ranged from 0.41 ±
0.06 to 0.43 ± 0.04 ng/ml, and 0.42 ± 0.04 to 0.44 ± 0.05
ng/ml, respectively (Figure 2(b)).
3.3. Effect of CCK on Bile Duct Contraction
Bile duct contraction in response to graded doses of
CCK was measured in an organ bath as described. Bile
duct rings obtained from normal rats contracted in re-
sponse to CCK in a dose dependent manner. At 10-5 M
CCK, a 20% increase of the ring tension was observed
(Figure 3).
Bile duct rings prepared form hyperglycemic rats at 8
months after confirmation of hyperglycemia symptom
exhibited lower contractile tension under the same CCK
dose range (Figure 3). The contractile response to CCK
was somewhat dose responsive, however, at the highest
CCK concentration (10-5 M) used, the tension generated
was still lower than that of the normal without CCK
stimulation.
3.4. Expression of CCK Receptor in Bile
Duct
Total bile duct tissue lysates were prepared from normal
and hyperglycemicrats. Proteins were fractionated on a
10% SDS-PAGE, transferred to cellulose nitrate mem-
brane and blotted for CCK-A receptor. Figure 4 is a rep-
(a)
(b)
Figure 2. Plasma CCK levels in STZ-nicotinamide induced
hyperglycemic rats. (a) during fed stage; (b) during fast stage.
Data are presented as means ± SEM, n value was 8 for each
experimental group. ■, Normal SD rats; □, STZ-nicotinamide
induced hyperglycemic rats.
resentative blot shown that the protein content of the
CCK-A receptor in hyperglycemic rats was reduced by
about 50% compared to that of the normal rats.
4. DISCUSSION
Epidemiological studies have shown that obesity, hyper-
insulinemia, and diabetes are important risk factors for
the development of the gallstones [23,24]. Hyperinsu-
linemia, or insulin resistance, and obesity are among the
metabolic syndrome cluster, and people with metabolic
syndrome have been shown to predispose to diabetes,
cardiovascular disease, hypertension and gallstone dis-
ease [25]. Metabolic disorders may affect the biochemi-
cal characteristics, especially the lipid composition of the

C.-M. Liu et al. / HEALTH 2 (2010) 1072-1077
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1076
Figure 3. Dose effect of CCK on the contraction of bile duct
isolated from normal (■) and STZ-nicotinamide induced hy-
perglycemic rats (▲). Bile duct of the STZ-nicotinamide in-
duced hyperglycemic rats was isolated at 8 months post iduc-
tion.
Figure 4. CCK-A receptor protein expression in the bile duct
of the normal and hyperglycemic rats.
bile. In fact, cholesterol supersaturation or an increase
cholesterol to phospholipids ratio has been suggested to
be one of the determinants for cholesterol gallstone for-
mation [26]. One other possible influencing factor is the
contractile function of the gallbladder. The major hor-
monal regulator of gallbladder contraction in the intesti-
nal phase of digestive function is CCK [27,28]. Reduced
gallbladder contraction function due to defect in CCK
signaling has been shown to impair gallbladder empting
and enhance gallstone formation [29]. From a recent
study we know, gallbladder dysmotility may have accel-
erated sludge and gallstone formation in male and fe-
male CCK-1(A) receptor-deficient mice, but its contri-
bution was limited [30]. Since diabetes patients, which
are characterized by chronic hyperglycemia have been
reported to have a higher incidence of gallbladder stone
disease, we therefore, sought to examine the possible
effects of the hyperglycemia on the gallbladder contrac-
tile function in response to CCK in rats.
We found that bile duct contractility of the chronically
hyperglycemia rats with and without CCK stimulation
was consistently lower that of the normal rats. Further
examination by Western blotting showed that the ex-
pression of CCK-A receptor protein in the bile duct of
the hyperglycemic rats was reduced by 50% compared
with that of the normal rats. It is therefore concluded that
reduced bile duct response to CCK contractile stimula-
tion in hyperglycemic, and perhaps, diabetic rats is due,
at least in part, to a reduced expression of CCK-A re-
ceptor. Interestingly, the plasma CCK contents of the
normal and hyperglycemic rats were not significantly
differed. Thus, the reduced bile duct contractility may be
one of the important influencing factors of the high co-
incidence of gallbladder stone disease and diabetes.
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