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Comparative Biochemistry and Physiology, Part C 143 (2006) 429 – 435

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Effect of ethanolic extracts of Ananas comosus L. leaves on insulin


sensitivity in rats and HepG2
Weidong Xie a , Wei Wang b , Hui Su b , Dongming Xing b , Yang Pan b , Lijun Du b,⁎
a
Life Sciences Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
b
Laboratory of Pharmaceutical Sciences, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
Received 5 August 2005; received in revised form 20 March 2006; accepted 13 April 2006
Available online 28 April 2006

Abstract

Ethanolic extracts of Ananas comosus L. leaves (AC) enriched with phenols have hypoglycemic activity in diabetic rats. Here, we investigated
the effect of AC on insulin sensitivity in rats and HepG2. In high-fat diet-fed and low-dose streptozotozin-treated diabetic Wistar rats subjected to
challenge with exogenous human insulin, AC treatment at an oral dose of 0.40 g/kg could significantly improve sensitivity to exogenous insulin.
After a sub-acute treatment, AC also could inhibit the development of insulin resistance in high-fat diet-fed and low-dose streptozotozin-treated
diabetic rats following the test of loss of tolbutamide-induced blood glucose lowering action. For intravenous insulin/glucose infusion test, high-
fat diet-fed and low-dose alloxan-treated Wistar rats were associated with insulin resistance, which was improved after AC or fenofibrate
treatment. AC application inhibited the development of insulin resistance in HepG2 cells. The above animal models were well developed to
simulate type 2 diabetes. Taken together, our results suggest that AC may improve insulin sensitivity in type 2 diabetes and could be developed
into a new potential natural product for handling of insulin resistance in diabetic patients.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Ananas comosus L.; Pineapple; Fenofibrate; Insulin resistance; Metformin; Tolbutamide; Type 2 diabetes; Glycogen; High-fat diets

1. Introduction for type 2 diabetes (Scheen, 2004). Type 2 diabetes is also


produced in animal models derived from genetic and environ-
Insulin is an important hormone in nutrient metabolism. Due mental factors (Trevaskis et al., 2004; Chen and Wang, 2005;
to the remarkable therapeutic effect of purified and synthetic Reed et al., 2000) that are usually used to simulate type 2 diabetic
insulin, natural products were gradually abandoned in many patients, to study their pathophysiological characters, and to
nations and areas. However, complications in macrovascular or evaluate the actions or their mechanisms of the tested agents.
microvascular functions are still associated in the patients re- Ananas comosus L. (Bromeliaceae) has long been one of the
ceiving insulin injection (Egger et al., 1997). Insulin resistance is most popular of tropical and subtropical fruits. It is grown
another serious clinical problem associated with hypertension, extensively in Hawaii, Philippines, Caribbean area, Malaysia,
type 2 diabetes, dyslipidemia, coronary artery disease, obesity, Taiwan, Thailand, Australia, Mexico, Kenya, South Africa and
abnormal glucose tolerance etc. (Cordain et al., 2003; Kopel- Hainan province of China. Besides agricultural utilities such as
man, 2004). Insulin resistance, defined as impaired insulin-me- the fruits for nutritional food, some folk medicinal uses were
diated glucose disposal, is a common consequence of excess found. In Thailand, A. comosus was used as an indigenous
body weight and cause of impaired glucose tolerance in type 2 medicinal plant (Sripanidkulchai et al., 2000, 2001) for the
diabetes (Mensah et al., 2004). Recent decades have seen a treatzment of dysuria. In China, A. comosus cortexes served as
resurgent interest in the development of insulin sensitizing agent alexiphamic, antitussive and antidiarrhea agents and A. comosus
leaves were usually used as antidyspepsia or antidiarrhea agents
in Chinese Traditional Medicine (Song, 1999).
⁎ Corresponding author. Tel./fax: +86 10 62773630. Considering that the values to traditional medicine of A.
E-mail address: pharm@mail.tsinghua.edu.cn (L. Du). comosus leaves and since no systematic scientific studies have
1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2006.04.002
430 W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

been carried out on them we decided to further develop A. 2.3. Collection of plant material
comosus leaves for medicinal uses, that may benefit people of
any socio-economic class. In our preliminary study, the ethanolic Leaves of Ananas comosus L. were collected from Boao,
extracts of A. comosus L. leaves (AC) have anti-diabetic effects Hainan province of China and authenticated by Dr. Shouquan Lin,
(Xie et al., 2005a) in diabetic dyslipidemic rats induced by the Institute of Medicinal Plant, Chinese Academy of Medical
alloxan and high-fat diets. However, AC has no hypoglycemic Sciences. AVoucher specimen (No. 020501) was deposited in the
effect in normal rats. Therefore, AC may exert a hypoglycemic herbarium of Laboratory of Pharmaceutical Sciences, Department
effect by promoting peripheral utilization of glucose or of Biological Sciences and Biotechnology, Tsinghua University.
enhancing the sensitivity of insulin in diabetic animals. In this This plant was dried in shade, powdered and the powder was used
study, we investigated the effect of AC on insulin sensitivity in for the ethanolic extraction.
rats and HepG2. STZ or alloxan-treated and high-fat diet-fed
Wistar rats were selected as diabetic animal models because of 2.4. Preparation of extract
the close similarities with diabetic patients (Zhang et al., 2003;
Xie et al., 2005a). HepG2 cells were used in this study due to Dried powder of A. comosus leaves was refluxed with 70%
their common physiological function to lipid or glucose ethanol three times for 1 h. Each extracted solution was
metabolism with normal hepatic cells (Xu et al., 2003). condensed to a final concentration, 0.2 g/ml (in terms of dried
starting material). Then, the extracted suspension was static for
2. Materials and methods 10 h in the dark and shade and then the supernatant was used to
elute through a resin column (HPD-100, China). Firstly, it was
2.1. Materials eluted with distilled water until no sugar was detected (the eluted
water was more than 5 times volume of the column). Sub-
HepG2 cells were provided by the Cell Bank of the Institute of sequently, 80% ethanol was used to elute the column and the
Fundamental Medicine, Chinese Academy of Medical Science elute was collected, condensed and dried to the extract for
(Beijing, China). Bovine serum albumin (BSA), fetal bovine experimental uses. The yield of the ethanolic extract of A.
serum (FBS), Dulbecco's modified Eagle's medium (DMEM) comosus leaves (AC) was 3.3% (w/w in terms of dried starting
were obtained from GIBCO. Biosynthetic human insulin was material). Phytochemical screening of the ethanolic extract con-
supplied by Novo Nordisk Pharmaceutical Industries, Inc. (North tained total phenols (60%, w/w in terms of the extract). Known
Carolina, USA). Streptozotozin (STZ) and alloxan were purchased phenols included P-coumaric acid (1.5%), 1-O-P-coumaroylgly-
from Sigma Chemical Co. (St. Louis, MO, USA). Blood glucose cerol (0.3%), caffeic acid (1.0%) and 1-O-caffeoylglycerol
diagnosis kits were obtained from ZhongSheng Beikong High- (0.2%).
tech Engineering Co. (Beijing, China). Metformin and tulbotamide
were supplied by Beijing Medicinal Company (Beijing, China). 2.5. Effects of ethanolic extracts of Ananas comosus L. leaves
Coommassie brilliant blue (G250) was purchased from Xinjingke on insulin sensitivity in low-dose STZ-treated and high-fat diet-
Co. Ltd. (Beijing, China). Final concentrations of Dimethyl Sul- fed Wistar rats
phoxide (DMSO) for using for dissolving drugs in medium were
below 0.1% (v/v). Other chemicals were all reagent grade. Diabetes was induced in male Wistar rats by intraperitoneal
(ip) injection of a low-dose STZ (30 mg/kg/day, once a day,
2.2. Animals and diets continuous for 2 days), freshly dissolved in citrate buffer
(0.01 mol/l, pH 4.5) as outlined by Milani et al. (2005) with a
Male Wistar rats (Rattus norvegicus 180–190 g) were ob- slight modification; simultaneously high-fat diets were fed to
tained from the Laboratory Animal Institute of Chinese Aca- these rats. This diabetic model is associated with insulin resistance
demy of Medical Science (Beijing, China). Animals were kept (Reed et al., 2000). Two weeks later, rats with marked hy-
in an environmentally controlled breeding room (temperature: perglycaemia were selected for the study. Rats were used to
20 ± 2 °C, humidity: 60 ± 5%, 12 h dark/light cycle). They were investigate the response to exogenous insulin. Diabetic rats were
fed standard laboratory chow with water ad libitum and fasted divided into five groups of six rats in each group: diabetic rats
overnight before the experiments. Rats were maintained in treated with AC at a dose of 0.80 g/kg; diabetic rats treated with
accordance with internationally accepted principles for labora- AC at a dose of 0.40 g/kg; diabetic rats treated with AC at a dose
tory animal use. Normal and high-fat diets were obtained from of 0.20 g/kg; diabetic rats treated with metformin at a dose of
the Institute of Experimental Animals, Chinese Academy of 0.32 g/kg; diabetic controls. Rats fasting overnight were orally
Medical Sciences. Components of the diets were described in administrated AC at different doses. After 2 h of administration,
our previous study (Xie and Du, 2005). The normal chow diets they were used to challenge with exogenous insulin according to
contained 20% crude protein, 4% crude fat, 5% crude fiber, 8% the previous method (Wu et al., 2002) with slight modifications.
crude ash, 1.2% calcium, 0.6% phosphorous and 54% nitrogen In brief, an intravenous insulin challenged test was performed by
free extract. High-fat diets contained 5% lard, 3% cholesterol, giving 0.2 U/kg of short-acting human insulin into these rats.
0.3% sodium deoxycholate and 91.7% normal chow diets. In Blood samples from the femoral vein were drawn for measuring
addition, for high-fat diet fed rats, soluble lard was also fed to blood glucose concentrations at 0 and 1 h following the intra-
these rats at a fixed dose of 10 ml/kg/d. venous insulin challenge test.
W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435 431

2.6. Effect of ethanolic extracts of Ananas comosus L. leaves was controlled at a flow-rate of 10 μl//kg/min. Blood samples were
on insulin resistance in low-dose STZ-treated and high-fat diet- taken for arterial glucose at 10-min intervals over 40 min.
fed Wistar rats Following 40 min of insulin infusion (40 μl/kg/min) and 30 min
of glucose infusion (10 μl/kg/min), the rats were allowed stabilized
Insulin resistance was induced in male Wistar rats as prescribed until three successive stable glucose concentrations were obtained
above. Animals were divided into six groups of six rats in each as described above. The mean of three successive stable glucose
group: tolbutamide-treated both normal and diabetic rats; AC- concentrations was regarded as basal glucose level. From then on,
treated both normal and diabetic ones; tolbutamide/AC-treated glucose infusion rate was adjusted to 20, 40, and 60 μl/kg/min for
both normal and diabetic ones. AC-treated both normal and dia- 10-min intervals, respectively. Blood samples were taken for ar-
betic rats, and tolbutamide/AC-treated both normal and diabetic terial glucose assays at 10-min intervals over 30 min. Data were
ones, received 15 days treatment of AC at a dose of 0.40 g/kg/d expressed as percentage % increase over basal glucose level.
while tolbutamide-treated both normal and diabetic rats received
identical volumes of distilled water. Insulin resistance was iden- 3. Cell culture
tified using the loss of tolbutamide-induced plasma glucose lo-
wering action as described previously (Chi et al., 1998). In brief, 3.1. Insulin-resistant HepG2 cell model
rats under sodium pentobarbital (50 mg/kg, ip) anesthesia were
received an ip injection of 10 mg/kg tolbutamide 8 h after the HepG2 were seeded into 96 multi-well plates in DMEM sup-
treatment with ethanolic extracts. Tolbutamide-treated both normal plemented with 10% fetal bovine serum (FBS) and penicillin/
and diabetic rats, and tolbutamide/AC-treated both normal and streptomycin (100 U/ml each), and cultured in a humidified incu-
diabetic ones received tolbutamide injection while AC-treated bator (5% CO2) at 37 °C. The cells were allowed to attach for at
both normal and diabetic rats received identical volumes of ve- least 24 h. Insulin-resistant cell model was induced according to the
hicles. Blood glucose levels were determined using blood samples previous method (Li et al., 1999) with a slight modification. In
collected from femoral veins of rats at 1 h after tolbutamide brief, HepG2 cells were incubated with fresh medium containing
injection. 1% FBS, 10− 7 M human insulin and AC (0.1, 1, 10 and 100 μg/ml,
respectively) or metformin (10 μg/ml) for 24 h. Subsequently, the
2.7. Intravenous insulin/glucose infusion test medium is exchanged with medium containing 1% FBS, 10− 9 M
insulin and 5–15 mM glucose; incubation was conducted in this
Diabetic dyslipidemic model was induced in male Wistar rats medium for 12–24 h.
weighing 180–200 g by a high-fat diet fed for 8 weeks and alloxan
treated (ip, 80 mg/kg) at the 2nd and 3rd week of a high-fat diet 3.2. Extracellular glucose and intracellular glycogen in HepG2
fed. Simultaneously, high-fat diet-fed animals were also orally
administrated the tested agents. Animals were divided into four The compounds at these ranges did not influence cellular
groups: untreated normal controls; untreated diabetic dyslipi- bioactivity. After treatment, the following assays were started.
demic controls; fenofibrate-treated diabetic dyslipidemic rats 10 μl of medium was assayed glucose by enzymatic methods.
(0.20 g/kg/d); AC-treated diabetic dyslipidemic rats (0.40 g/kg/d). Data were expressed as consumption of extracellular glucose
At the 8th week, exogenous insulin/glucose infusion test was content (nmol/mg protein) = [extracellular glucose content
performed in rats weighing 380–400 g. Rats did not receive the test (nmol)0 h − extracellular glucose content (nmol)12–24 h] / mg cell
agents prior to 12 h of the experiment. The rats were not fasted and protein. The mass of intracellular glycogen was measured as
were anesthetized with an ip injection of 10% Urethane (g/100 ml) prescribed previously (Gomez-Lechon et al., 1996). Cell protein
solution at a dose of 10 ml/kg. Rats were placed on their back and
heparinized with heparin (50 U/kg). Heparinized polyethylene tube
was inserted into the left jugular artery where blood samples (25 μl) Table 1
were collected for arterial glucose analysis. Another heparinized Changes of plasma glucose concentration and the blood glucose lowering
polyethylene tube was inserted into the left femoral vein. The end activity of insulin in diabetic Wistar rats induced by STZ and high-fat diets
of the heparinized polyethylene tube was tightly connected to a Groups Dosage Blood glucose Blood glucose lowering
three-way connector. Two heparinized polyethylene tubes were (g/kg) (mmol/l) activity of insulin %
connected to the three-way connector, one for 10% glucose infu- At 0 h At 1 h
sion (g/ml), and the other for 1 U/ml human insulin infusion, which AC 0.80 11.0 ± 0.4 7.3 ± 1.1⁎ 42.5 ± 5.4
were controlled by two variable infusion pumps, respectively. 0.40 11.6 ± 1.4 4.5 ± 0.8⁎⁎ 61.5 ± 4.4##
The rats were allowed to stabilize after surgery. Subsequently, 0.20 10.8 ± 2.6 6.5 ± 2.7 48.0 ± 7.6
arterial blood samples were taken at 5-min intervals and arterial Metformin 0.32 12.4 ± 2.2 8.0 ± 2.0 50.3 ± 4.7##
Control 11.3 ± 2.0 8.0 ± 1.5 29.2 ± 2.9
blood glucose concentrations were immediately analyzed with a
glucose analyzer. When three successive stable glucose concentra- Data were expressed as Mean ± SD (n = 6). ⁎P b 0.05, ⁎⁎P b 0.01, compared blood
tions were obtained, the mean of three glucose concentrations was glucose levels at between 0 h and 1 h in each group by ANOVA; ##P b 0.01. Prior
to statistical analysis, percentages (p) were conducted the transformation of arc
referred to as basal glucose level. Then, insulin infusion started; its sin and square root, i.e. y = arc sin1/2 p, and then compared with control group by
flow-rate was controlled at 40 μl/kg/min all over the course. ANOVA followed by Newman–Keuls test. “AC”, the ethanolic extracts of
Following 10 min of insulin infusion, glucose infusion started and Ananas comosus L. leaves.
432 W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

Table 2 Without AC treatment, there was a loss of tolbutamide-induced


Changes of plasma glucose concentration and the plasma glucose lowering blood glucose lowering action in tolbutamide-treated diabetic
activity of tolbutamide (10 mg/kg, ip) in normal and diabetic Wistar rats induced
by STZ and high-fat diets
rats (no significant fall in blood glucose) while this phenomenon
did not occur in tolbutamide-treated normal ones [(20.7 ± 2.5) %
Groups Blood glucose (mmol/l) Blood
glucose
fall] (Table 2), indicating insulin resistance in diabetic rats. After
At 0 h At 1 h treatment with AC, tolbutamide-induced blood glucose was
lowering
activity % significantly lowered in tolbutamide/AC-treated diabetic rats
Tolbutamide-treated normal rats 8.7 ± 0.7 6.9 ± 0.8⁎⁎ 20.7 ± 2.5# [(27.0 ± 3.4) % fall]. There are also a significant decrease in
AC-treated normal rats 7.9 ± 0.5 7.6 ± 0.2 4.3 ± 1.4 tolbutamide/AC-treated normal rats [(20.1 ± 1.9) % fall]. How-
Tolbutamide /AC-treated normal rats 8.2 ± 0.4 6.6 ± 0.4⁎⁎ 20.1 ± 1.9# ever, AC had no significant decrease in blood glucose in AC-
Tolbutamide-treated diabetic rats 11.4 ± 0.7 11.0 ± 2.1 7.3 ± 3.3 treated normal and diabetic rats not exposed to tolbutamide.
AC-treated diabetic rats 9.6 ± 0.8 9.4 ± 1.2 3.3 ± 1.9
Tolbutamide/AC-treated diabetic rats 9.8 ± 0.6 7.1 ± 0.7⁎⁎ 27.0 ± 3.4##
Data were expressed as Mean±SD (n=6), ⁎⁎P b 0.01 compared with blood glucose
levels at between 0 h and 1 h in each group by ANOVA. #P b 0.05, ##P b 0.01, Prior to
statistical analysis, percentages (p) were conducted the transformation of arc sin and
square root, i.e. y =arc sin1/2 p, and then compared with tolbutamide-treated diabetic
rats (Group IV) by ANOVA followed by Newman–Keuls test. “AC”, ethanolic
extracts of Ananas comosus L. leaves at an oral dose of 0.4 g/kg.

was solubilized in 0.1–0.5 N NaOH and quantified with Co-


omassie blue G-250 by the Bradford technique (Fanger, 1987).

3.3. Statistical analyses

All values were expressed as mean ± SD. Data were sta-


tistically analyzed by analysis of variance (ANOVA). The New-
man–Keuls comparisons were used to determine the source of
significant differences where appropriate. P values below 0.05
were considered statistically significant. In addition, prior to
statistical analysis, percentages will be conducted the transfor-
mation of arc sin and square root, i.e., y = arc sin1/2 p.

4. Results

4.1. Effects of Ananas comosus L. leaf extract on insulin


sensitivity in diabetic rats

At 1 h of treatment of insulin, a significant lowered blood


glucose level was observed in diabetic rats treated with both AC
at three different doses (0.80, 0.40 and 0.20 g/kg) and metformin
(0.32 g/kg) compared with that at 0 h (Table 1). At 1 h of
treatment of insulin, a further decrease in blood glucose was
produced in diabetic rats treated with AC at a dose of 0.40 g/kg
as well as tolbutamide at a dose of 0.32 g/kg compared with
untreated controls. A dose of 0.4 g/kg AC achieved the largest Fig. 1. Changes in blood glucose levels in normal and diabetic dyslipidemic
effect. Therefore, this dosage of AC was adopted in the Wistar rats following intravenous insulin/glucose infusion. (a) Insulin infusion at
following trials. No significant difference was observed between the flow-rate of 40 μl/kg/min for 40 min, following 10 min of insulin infusion,
AC and metformin treated diabetic rats in the present study. glucose infusion at the flow-rate of 10 μl/kg/min for 30 min; (b) insulin infusion
at the flow-rate of 40 μl/kg/min, glucose infusion at the flow-rate of 10, 20, 40
and 60 μl/kg/min for 10-min intervals. “DC” diabetic dyslipidemic control
4.2. Effect of Ananas comosus L. leaf extract on insulin group; “AC”, the diabetic dyslipidemic group treated with the ethanolic extracts
resistance in diabetic rats of Ananas comosus L. leaves at a dose of 0.4 g/kg. “NC”, normal control group;
“FB”, the diabetic dyslipidemic group treated with fenofibrate at a dose of 0.2 g/
At 1 h of treatment of tolbutamide, a significant decrease in kg. Data were expressed as percentage % increase over basal glucose level
(n = 4–5). Prior to statistical analysis, percentages ( p) were conducted the
blood glucose levels was observed in tolbutamide-treated nor- transformation of arc sin and square root, i.e. y = arc sin1/2 p. aP b 0.05, aaP b 0.01,
mal rats, tolbutamide/AC-treated normal ones, and tolbutamide/ AC vs. DC; bP b 0.05, bbP b 0.01, FB vs. DC; cP b 0.05, ccP b 0.01, NC vs. DC by
AC-treated diabetic ones compared with those at 0 h (Table 2). ANOVA followed by Newman–Keuls test.
W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435 433

4.3. Effect of Ananas comosus L. leaf extract on blood glucose 5. Discussion


in diabetic rats following insulin/glucose infusion
STZ or alloxan induced rats produced hyperglycemia. Both
During 40 min of insulin infusion, blood glucose level in compounds are widely used to mimic diabetic patients and to
untreated diabetic dyslipidemic control rats fell at a slower rate at evaluate hypoglycemic or related effects of compounds and
10, 20, 30 and 40 min of insulin infusion than in untreated normal extracts (Kordowiak et al., 2000; Pari and Saravanan, 2002).
controls (Fig. 1a), which indicated that insulin insensitivity was Single administration of AC could enhance exogenous insulin
produced in these rats. AC or fenofibrate-treated diabetic sensitivity in diabetic rats. However, we did not know if AC had a
dyslipidemic rats showed a more rapid decrease at 10, 30 and direct hypoglycemic effect in the diabetic rats. Interestingly, no
40 min of insulin infusion than untreated diabetic dyslipidemic significant change was observed in the diabetic rats when AC was
controls, indicating that AC or fenofibrate could enhance the separately used (without the injection of exogenous insulin) in rats
sensitivity to exogenous insulin. No significant difference was in an additional trial (data not shown). Therefore, this effect of AC
observed between AC and fenofibrate treated animals. might be associated with the promotion of insulin sensitivity, and
Following glucose infusion (Fig. 1b), untreated diabetic dy- not a result from a direct hypoglycemic effect of AC. Metformin,
slipidemic control rats produced a higher level of blood glucose believed to alleviate insulin resistance in the presence of insulin
than untreated normal controls at 20 and 30 min of variable (Bailey, 1993), was selected as positive control to evaluate
glucose infusion. This suggests that diabetic dyslipidemic rats effectiveness of the model. Metformin has been shown to improve
show impaired insulin function in glucose disposal. A smaller the insulin sensitivity in type 2 diabetes by activating post-recep-
increase in blood glucose level was shown in AC or fenofibrate- tor insulin signaling pathways. AC at a dose of 0.4 g/kg was more
treated diabetic dyslipidemic rats at 20 and 30 min than in effective than metformin (0.32 g/kg). To challenge with exogenous
untreated diabetic dyslipidemic controls. It appears that AC or insulin is an acute test used to evaluate effect of AC on improving
fenofibrate could enhance insulin function in glucose disposal. No insulin sensitivity and to determine the most effective dose in this
significant difference was observed between AC and fenofibrate trial. In the other sub-acute trials, we have adopted this most
treated animals. effective dose (0.4 g/kg). However, whether AC exerted the effect
by the action mechanism of metformin remained undetermined.
4.4. Effects of Ananas comosus L. leaf extract on sensitivity to Insulin resistance was associated with a loss of tolbutamide-
exogenous insulin in insulin-resistant HepG2 cells induced blood glucose lowering action (Chi et al., 1998; Chang
et al., 1999). Tolbutamide belongs to hypoglycemic sulphony-
Following 10− 9 M insulin incubation, of HepG2 cell there lureas to stimulate insulin release in diabetic patients. If patients
was a significant decrease in the consumption of extracellular show insulin resistance, they will show a loss of tolbutamide-
glucose in controls with 10− 7 M insulin pretreatment (P b 0.01) induced blood glucose lowering action. However, insulin sen-
compared with controls without insulin pretreatment (Table 3). sitizer, thiazolidinediones, may strengthen the hypoglycaemic
Insulin at the final concentration of 10− 9 M significantly in- effect of tolbutamide. Tolbutamide had no significant effect in
creased this lowered consumption of extracellular glucose in diabetic rats in the present trial. It indicates these diabetic rats
HepG2 cells pretreated with 10− 7 M insulin combined with might be associated with insulin resistance. Our results showed
metformin (10− 5 g/ml) or AC (10− 5, 10− 6, 10− 7 g/ml), re- that AC enhanced tolbutamide-induced blood glucose lowering
spectively. Intracellular glycogen was decreased in AC (10− 5 action, and then inhibited the development of insulin resistance
and 10− 6 g/ml)-treated HepG2 cells. No change was noted in in insulin resistant rats induced by low-dose STZ and high-fat
metformin-treated HepG2 cells. The effect of AC at 10− 5 g/ml diets. These results suggest that AC can enhance insulin sen-
was comparable to that of metformin at 10− 5 g/ml. sitivity. In addition, if AC could stimulate insulin secretion, it
should have a hypoglycaemic effect in normal rats because their
insulin release is in good condition, e.g., tolbutamide had a
Table 3
Effect of the ethanolic extracts of Ananas comosus L. leaves (AC) on sensitivity
hypoglycaemic effect in normal rats. Interestingly, no signifi-
to exogenous insulin in insulin-resistant HepG2 cells pretreated with 10−7 M cant effect was observed in normal rats when AC was separately
insulin used without the administration of tolbutamide, further sug-
Groups Dosage Consumption of Intracellular glycogen gesting the idea that AC affects insulin sensitivity, and does not
(g/ml) extracellular glucose (μg/mg cell protein) play a direct hypoglycaemic role. It seems that AC exerts a
(nmol/mg cell protein) hypoglycaemic effect by enhancing insulin sensitivity not by
Blank control 49.08 ± 1.73 0.190 ± 0.012 stimulating insulin secretion. However, the precise mechanisms
Control 46.79 ± 1.53## 0.204 ± 0.018 of AC need the further investigation.
Metformin 10−5 50.75 ± 1.07⁎⁎ 0.214 ± 0.016 Following intravenous insulin and glucose infusion test,
AC 10−4 47.44 ± 0.55 0.185 ± 0.023
insulin resistance was also produced in low-dose alloxan-treated
10−5 49.77 ± 0.83⁎⁎ 0.166 ± 0.010⁎⁎
10−6 49.28 ± 1.82⁎ 0.180 ± 0.015⁎ and high-fat diet fed rats, which may become a kind of new animal
10−7 48.34 ± 0.26⁎ 0.188 ± 0.010 model to simulate type 2 diabetes. Here, the reason why we used
Data were expressed as Mean ± SD (n = 5). ##P b 0.01 vs. blank control without alloxan instead of STZ was because not only alloxan was cheaper
10−7 M insulin pretreatment by ANOVA; ⁎P b 0.05, ⁎⁎P b 0.01 vs. control with than STZ but also it worked as well as STZ in the preliminary
10−7 M insulin pretreatment by ANOVA followed by Newman–Keuls test. study. AC significantly lowered blood glucose following insulin
434 W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

infusion compared with control, suggesting that AC improves comosus leaves on diabetic rats and to determine its active
exogenous insulin sensitivity. Following glucose infusion, a compounds.
smaller increase in blood glucose was observed in diabetic rats,
suggesting that AC enhances the insulin function in glucose Acknowledgments
disposal in diabetic rats. The increase in glucose disposal of AC
may be not related to stimulate insulin secretion because: AC had The study was supported by the Science and Development
no effect in normal rats; endogenous insulin was much less than Foundation Tsinghua University (No. A2005568).
exogenous insulin and was neglectable in diabetic rats. AC
ameliorated insulin sensitivity in this model, a result that is con- References
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