gy, Part C 143 (2006) 429–435www.elsevier.com/locate/cbpc

Comparative Biochemistry and Physiolo

Effect of ethanolic extracts of Ananas comosus L. leaves on insulinsensitivity 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, Chinab 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 2006Available online 28 April 2006

Abstract

Ethanolic extracts of Ananas comosus L. leaves (AC) enriched with phenols have hypoglycemic activity in diabetic rats. Here, we investigatedthe effect of AC on insulin sensitivity in rats and HepG2. In high-fat diet-fed and low-dose streptozotozin-treated diabetic Wistar rats subjected tochallenge 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-treateddiabetic 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 fenofibratetreatment. AC application inhibited the development of insulin resistance in HepG2 cells. The above animal models were well developed tosimulate type 2 diabetes. Taken together, our results suggest that AC may improve insulin sensitivity in type 2 diabetes and could be developedinto 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

Insulin is an important hormone in nutrient metabolism. Dueto the remarkable therapeutic effect of purified and syntheticinsulin, natural products were gradually abandoned in manynations and areas. However, complications in macrovascular ormicrovascular functions are still associated in the patients re-ceiving insulin injection (Egger et al., 1997). Insulin resistance isanother serious clinical problem associated with hypertension,type 2 diabetes, dyslipidemia, coronary artery disease, obesity,abnormal glucose tolerance etc. (Cordain et al., 2003; Kopel-man, 2004). Insulin resistance, defined as impaired insulin-me-diated glucose disposal, is a common consequence of excessbody weight and cause of impaired glucose tolerance in type 2diabetes (Mensah et al., 2004). Recent decades have seen aresurgent interest in the development of insulin sensitizing agent

⁎ Corresponding author. Tel./fax: +86 10 62773630.E-mail address: pharm@mail.tsinghua.edu.cn (L. Du).

1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpc.2006.04.002

for type 2 diabetes (Scheen, 2004). Type 2 diabetes is alsoproduced in animal models derived from genetic and environ-mental factors (Trevaskis et al., 2004; Chen and Wang, 2005;Reed et al., 2000) that are usually used to simulate type 2 diabeticpatients, to study their pathophysiological characters, and toevaluate the actions or their mechanisms of the tested agents.

Ananas comosus L. (Bromeliaceae) has long been one of themost popular of tropical and subtropical fruits. It is grownextensively in Hawaii, Philippines, Caribbean area, Malaysia,Taiwan, Thailand, Australia, Mexico, Kenya, South Africa andHainan province of China. Besides agricultural utilities such asthe fruits for nutritional food, some folk medicinal uses werefound. In Thailand, A. comosus was used as an indigenousmedicinal plant (Sripanidkulchai et al., 2000, 2001) for thetreatzment of dysuria. In China, A. comosus cortexes served asalexiphamic, antitussive and antidiarrhea agents and A. comosusleaves were usually used as antidyspepsia or antidiarrhea agentsin Chinese Traditional Medicine (Song, 1999).

Considering that the values to traditional medicine of A.comosus leaves and since no systematic scientific studies have

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.comosus leaves for medicinal uses, that may benefit people ofany socio-economic class. In our preliminary study, the ethanolicextracts of A. comosus L. leaves (AC) have anti-diabetic effects(Xie et al., 2005a) in diabetic dyslipidemic rats induced byalloxan and high-fat diets. However, AC has no hypoglycemiceffect in normal rats. Therefore, AC may exert a hypoglycemiceffect by promoting peripheral utilization of glucose orenhancing the sensitivity of insulin in diabetic animals. In thisstudy, we investigated the effect of AC on insulin sensitivity inrats and HepG2. STZ or alloxan-treated and high-fat diet-fedWistar rats were selected as diabetic animal models because ofthe close similarities with diabetic patients (Zhang et al., 2003;Xie et al., 2005a). HepG2 cells were used in this study due totheir common physiological function to lipid or glucosemetabolism with normal hepatic cells (Xu et al., 2003).

2. Materials and methods

2.1. Materials

HepG2 cells were provided by the Cell Bank of the Institute ofFundamental Medicine, Chinese Academy of Medical Science(Beijing, China). Bovine serum albumin (BSA), fetal bovineserum (FBS), Dulbecco's modified Eagle's medium (DMEM)were obtained from GIBCO. Biosynthetic human insulin wassupplied by Novo Nordisk Pharmaceutical Industries, Inc. (NorthCarolina, USA). Streptozotozin (STZ) and alloxan were purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). Blood glucosediagnosis kits were obtained from ZhongSheng Beikong High-tech Engineering Co. (Beijing, China). Metformin and tulbotamidewere supplied by Beijing Medicinal Company (Beijing, China).Coommassie brilliant blue (G250) was purchased from XinjingkeCo. Ltd. (Beijing, China). Final concentrations of Dimethyl Sul-phoxide (DMSO) for using for dissolving drugs in medium werebelow 0.1% (v/v). Other chemicals were all reagent grade.

2.2. Animals and diets

Male Wistar rats (Rattus norvegicus 180–190 g) were ob-tained from the Laboratory Animal Institute of Chinese Aca-demy of Medical Science (Beijing, China). Animals were keptin an environmentally controlled breeding room (temperature:20±2 °C, humidity: 60±5%, 12 h dark/light cycle). They werefed standard laboratory chow with water ad libitum and fastedovernight before the experiments. Rats were maintained inaccordance with internationally accepted principles for labora-tory animal use. Normal and high-fat diets were obtained fromthe Institute of Experimental Animals, Chinese Academy ofMedical Sciences. Components of the diets were described inour previous study (Xie and Du, 2005). The normal chow dietscontained 20% crude protein, 4% crude fat, 5% crude fiber, 8%crude ash, 1.2% calcium, 0.6% phosphorous and 54% nitrogenfree extract. High-fat diets contained 5% lard, 3% cholesterol,0.3% sodium deoxycholate and 91.7% normal chow diets. Inaddition, for high-fat diet fed rats, soluble lard was also fed tothese rats at a fixed dose of 10 ml/kg/d.

2.3. Collection of plant material

Leaves of Ananas comosus L. were collected from Boao,Hainan province ofChina and authenticated byDr. Shouquan Lin,the Institute of Medicinal Plant, Chinese Academy of MedicalSciences. AVoucher specimen (No. 020501) was deposited in theherbarium of Laboratory of Pharmaceutical Sciences, Departmentof Biological Sciences and Biotechnology, Tsinghua University.This plant was dried in shade, powdered and the powder was usedfor the ethanolic extraction.

2.4. Preparation of extract

Dried powder of A. comosus leaves was refluxed with 70%ethanol three times for 1 h. Each extracted solution wascondensed to a final concentration, 0.2 g/ml (in terms of driedstarting material). Then, the extracted suspension was static for10 h in the dark and shade and then the supernatant was used toelute through a resin column (HPD-100, China). Firstly, it waseluted with distilled water until no sugar was detected (the elutedwater was more than 5 times volume of the column). Sub-sequently, 80% ethanol was used to elute the column and theelute was collected, condensed and dried to the extract forexperimental uses. The yield of the ethanolic extract of A.comosus leaves (AC) was 3.3% (w/w in terms of dried startingmaterial). Phytochemical screening of the ethanolic extract con-tained total phenols (60%, w/w in terms of the extract). Knownphenols included P-coumaric acid (1.5%), 1-O-P-coumaroylgly-cerol (0.3%), caffeic acid (1.0%) and 1-O-caffeoylglycerol(0.2%).

2.5. Effects of ethanolic extracts of Ananas comosus L. leaveson insulin sensitivity in low-dose STZ-treated and high-fat diet-fed Wistar rats

Diabetes was induced in male Wistar rats by intraperitoneal(ip) injection of a low-dose STZ (30 mg/kg/day, once a day,continuous for 2 days), freshly dissolved in citrate buffer(0.01 mol/l, pH 4.5) as outlined by Milani et al. (2005) with aslight modification; simultaneously high-fat diets were fed tothese rats. This diabeticmodel is associatedwith insulin resistance(Reed et al., 2000). Two weeks later, rats with marked hy-perglycaemia were selected for the study. Rats were used toinvestigate the response to exogenous insulin. Diabetic rats weredivided into five groups of six rats in each group: diabetic ratstreated with AC at a dose of 0.80 g/kg; diabetic rats treated withAC at a dose of 0.40 g/kg; diabetic rats treated with AC at a doseof 0.20 g/kg; diabetic rats treated with metformin at a dose of0.32 g/kg; diabetic controls. Rats fasting overnight were orallyadministrated AC at different doses. After 2 h of administration,they were used to challenge with exogenous insulin according tothe previous method (Wu et al., 2002) with slight modifications.In brief, an intravenous insulin challenged test was performed bygiving 0.2 U/kg of short-acting human insulin into these rats.Blood samples from the femoral vein were drawn for measuringblood glucose concentrations at 0 and 1 h following the intra-venous insulin challenge test.

Table 1Changes of plasma glucose concentration and the blood glucose loweringactivity of insulin in diabetic Wistar rats induced by STZ and high-fat diets

Groups Dosage(g/kg)

Blood glucose(mmol/l)

Blood glucose loweringactivity of insulin %

At 0 h At 1 h

AC 0.80 11.0±0.4 7.3±1.1⁎ 42.5±5.40.40 11.6±1.4 4.5±0.8⁎⁎ 61.5±4.4##

0.20 10.8±2.6 6.5±2.7 48.0±7.6Metformin 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

Data were expressed asMean±SD (n=6). ⁎Pb0.05, ⁎⁎Pb0.01, compared bloodglucose levels at between 0 h and 1 h in each group by ANOVA; ##Pb0.01. Priorto statistical analysis, percentages (p) were conducted the transformation of arcsin and square root, i.e. y=arc sin1/2 p, and then compared with control group byANOVA followed by Newman–Keuls test. “AC”, the ethanolic extracts ofAnanas comosus L. leaves.

431W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

2.6. Effect of ethanolic extracts of Ananas comosus L. leaveson insulin resistance in low-dose STZ-treated and high-fat diet-fed Wistar rats

Insulin resistance was induced in maleWistar rats as prescribedabove. Animals were divided into six groups of six rats in eachgroup: tolbutamide-treated both normal and diabetic rats; AC-treated both normal and diabetic ones; tolbutamide/AC-treatedboth normal and diabetic ones. AC-treated both normal and dia-betic rats, and tolbutamide/AC-treated both normal and diabeticones, received 15 days treatment of AC at a dose of 0.40 g/kg/dwhile tolbutamide-treated both normal and diabetic rats receivedidentical volumes of distilled water. Insulin resistance was iden-tified using the loss of tolbutamide-induced plasma glucose lo-wering action as described previously (Chi et al., 1998). In brief,rats under sodium pentobarbital (50 mg/kg, ip) anesthesia werereceived an ip injection of 10 mg/kg tolbutamide 8 h after thetreatment with ethanolic extracts. Tolbutamide-treated both normaland diabetic rats, and tolbutamide/AC-treated both normal anddiabetic ones received tolbutamide injection while AC-treatedboth normal and diabetic rats received identical volumes of ve-hicles. Blood glucose levels were determined using blood samplescollected from femoral veins of rats at 1 h after tolbutamideinjection.

2.7. Intravenous insulin/glucose infusion test

Diabetic dyslipidemic model was induced in male Wistar ratsweighing 180–200 g by a high-fat diet fed for 8weeks and alloxantreated (ip, 80 mg/kg) at the 2nd and 3rd week of a high-fat dietfed. Simultaneously, high-fat diet-fed animals were also orallyadministrated the tested agents. Animals were divided into fourgroups: untreated normal controls; untreated diabetic dyslipi-demic controls; fenofibrate-treated diabetic dyslipidemic rats(0.20 g/kg/d); AC-treated diabetic dyslipidemic rats (0.40 g/kg/d).

At the 8th week, exogenous insulin/glucose infusion test wasperformed in rats weighing 380–400 g. Rats did not receive the testagents prior to 12 h of the experiment. The rats were not fasted andwere anesthetized with an ip injection of 10% Urethane (g/100 ml)solution at a dose of 10 ml/kg. Rats were placed on their back andheparinizedwith heparin (50U/kg). Heparinized polyethylene tubewas inserted into the left jugular arterywhere blood samples (25μl)were collected for arterial glucose analysis. Another heparinizedpolyethylene tube was inserted into the left femoral vein. The endof the heparinized polyethylene tube was tightly connected to athree-way connector. Two heparinized polyethylene tubes wereconnected to the three-way connector, one for 10% glucose infu-sion (g/ml), and the other for 1 U/ml human insulin infusion, whichwere controlled by two variable infusion pumps, respectively.

The rats were allowed to stabilize after surgery. Subsequently,arterial blood samples were taken at 5-min intervals and arterialblood glucose concentrations were immediately analyzed with aglucose analyzer. When three successive stable glucose concentra-tions were obtained, the mean of three glucose concentrations wasreferred to as basal glucose level. Then, insulin infusion started; itsflow-rate was controlled at 40 μl/kg/min all over the course.Following 10 min of insulin infusion, glucose infusion started and

was controlled at a flow-rate of 10 μl//kg/min. Blood samples weretaken for arterial glucose at 10-min intervals over 40 min.

Following 40min of insulin infusion (40μl/kg/min) and 30minof glucose infusion (10 μl/kg/min), the rats were allowed stabilizeduntil three successive stable glucose concentrations were obtainedas described above. The mean of three successive stable glucoseconcentrations was regarded as basal glucose level. From then on,glucose infusion rate was adjusted to 20, 40, and 60 μl/kg/min for10-min intervals, respectively. Blood samples were taken for ar-terial glucose assays at 10-min intervals over 30 min. Data wereexpressed as percentage % increase over basal glucose level.

3. Cell culture

3.1. Insulin-resistant HepG2 cell model

HepG2 were seeded into 96 multi-well plates in DMEM sup-plemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml each), and cultured in a humidified incu-bator (5% CO2) at 37 °C. The cells were allowed to attach for atleast 24 h. Insulin-resistant cell model was induced according to theprevious method (Li et al., 1999) with a slight modification. Inbrief, HepG2 cells were incubated with fresh medium containing1%FBS, 10−7 M human insulin andAC (0.1, 1, 10 and 100 μg/ml,respectively) or metformin (10 μg/ml) for 24 h. Subsequently, themedium is exchanged with medium containing 1% FBS, 10−9 Minsulin and 5–15 mM glucose; incubation was conducted in thismedium for 12–24 h.

3.2. Extracellular glucose and intracellular glycogen in HepG2

The compounds at these ranges did not influence cellularbioactivity. After treatment, the following assays were started.10 μl of medium was assayed glucose by enzymatic methods.Data were expressed as consumption of extracellular glucosecontent (nmol/mg protein)= [extracellular glucose content(nmol)0 h−extracellular glucose content (nmol)12–24 h] /mg cellprotein. The mass of intracellular glycogen was measured asprescribed previously (Gomez-Lechon et al., 1996). Cell protein

Table 2Changes of plasma glucose concentration and the plasma glucose loweringactivity of tolbutamide (10 mg/kg, ip) in normal and diabetic Wistar rats inducedby STZ and high-fat diets

Groups Blood glucose (mmol/l) Bloodglucoseloweringactivity %

At 0 h At 1 h

Tolbutamide-treated normal rats 8.7±0.7 6.9±0.8⁎⁎ 20.7±2.5#

AC-treated normal rats 7.9±0.5 7.6±0.2 4.3±1.4Tolbutamide /AC-treated normal rats 8.2±0.4 6.6±0.4⁎⁎ 20.1±1.9#

Tolbutamide-treated diabetic rats 11.4±0.7 11.0±2.1 7.3±3.3AC-treated diabetic rats 9.6±0.8 9.4±1.2 3.3±1.9Tolbutamide/AC-treated diabetic rats 9.8±0.6 7.1±0.7⁎⁎ 27.0±3.4##

Data were expressed as Mean±SD (n=6), ⁎⁎Pb0.01 compared with blood glucoselevels at between 0 h and 1 h in each group byANOVA. #Pb0.05, ##Pb0.01, Prior tostatistical analysis, percentages (p) were conducted the transformation of arc sin andsquare root, i.e. y=arc sin1/2 p, and then compared with tolbutamide-treated diabeticrats (Group IV) by ANOVA followed by Newman–Keuls test. “AC”, ethanolicextracts of Ananas comosus L. leaves at an oral dose of 0.4 g/kg.

Fig. 1. Changes in blood glucose levels in normal and diabetic dyslipidemicWistar rats following intravenous insulin/glucose infusion. (a) Insulin infusion atthe flow-rate of 40 μl/kg/min for 40 min, following 10 min of insulin infusion,glucose infusion at the flow-rate of 10 μl/kg/min for 30 min; (b) insulin infusionat the flow-rate of 40 μl/kg/min, glucose infusion at the flow-rate of 10, 20, 40and 60 μl/kg/min for 10-min intervals. “DC” diabetic dyslipidemic controlgroup; “AC”, the diabetic dyslipidemic group treated with the ethanolic extractsof 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/kg. Data were expressed as percentage % increase over basal glucose level(n=4–5). Prior to statistical analysis, percentages (p) were conducted thetransformation of arc sin and square root, i.e. y=arc sin1/2 p. aPb0.05, aaPb0.01,AC vs. DC; bPb0.05, bbPb0.01, FB vs. DC; cPb0.05, ccPb0.01, NC vs. DC byANOVA followed by Newman–Keuls test.

432 W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

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 ofsignificant differences where appropriate. P values below 0.05were considered statistically significant. In addition, prior tostatistical 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 insulinsensitivity in diabetic rats

At 1 h of treatment of insulin, a significant lowered bloodglucose level was observed in diabetic rats treated with both ACat 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 oftreatment of insulin, a further decrease in blood glucose wasproduced in diabetic rats treated with AC at a dose of 0.40 g/kgas well as tolbutamide at a dose of 0.32 g/kg compared withuntreated controls. A dose of 0.4 g/kg AC achieved the largesteffect. Therefore, this dosage of AC was adopted in thefollowing trials. No significant difference was observed betweenAC and metformin treated diabetic rats in the present study.

4.2. Effect of Ananas comosus L. leaf extract on insulinresistance in diabetic rats

At 1 h of treatment of tolbutamide, a significant decrease inblood glucose levels was observed in tolbutamide-treated nor-mal rats, tolbutamide/AC-treated normal ones, and tolbutamide/AC-treated diabetic ones compared with those at 0 h (Table 2).

Without AC treatment, there was a loss of tolbutamide-inducedblood glucose lowering action in tolbutamide-treated diabeticrats (no significant fall in blood glucose) while this phenomenondid not occur in tolbutamide-treated normal ones [(20.7±2.5) %fall] (Table 2), indicating insulin resistance in diabetic rats. Aftertreatment with AC, tolbutamide-induced blood glucose wassignificantly lowered in tolbutamide/AC-treated diabetic rats[(27.0±3.4) % fall]. There are also a significant decrease intolbutamide/AC-treated normal rats [(20.1±1.9) % fall]. How-ever, AC had no significant decrease in blood glucose in AC-treated normal and diabetic rats not exposed to tolbutamide.

433W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

4.3. Effect of Ananas comosus L. leaf extract on blood glucosein diabetic rats following insulin/glucose infusion

During 40 min of insulin infusion, blood glucose level inuntreated diabetic dyslipidemic control rats fell at a slower rate at10, 20, 30 and 40min of insulin infusion than in untreated normalcontrols (Fig. 1a), which indicated that insulin insensitivity wasproduced in these rats. AC or fenofibrate-treated diabeticdyslipidemic rats showed a more rapid decrease at 10, 30 and40 min of insulin infusion than untreated diabetic dyslipidemiccontrols, indicating that AC or fenofibrate could enhance thesensitivity to exogenous insulin. No significant difference wasobserved between AC and fenofibrate treated animals.

Following glucose infusion (Fig. 1b), untreated diabetic dy-slipidemic control rats produced a higher level of blood glucosethan untreated normal controls at 20 and 30 min of variableglucose infusion. This suggests that diabetic dyslipidemic ratsshow impaired insulin function in glucose disposal. A smallerincrease in blood glucose level was shown in AC or fenofibrate-treated diabetic dyslipidemic rats at 20 and 30 min than inuntreated diabetic dyslipidemic controls. It appears that AC orfenofibrate could enhance insulin function in glucose disposal. Nosignificant difference was observed between AC and fenofibratetreated animals.

4.4. Effects of Ananas comosus L. leaf extract on sensitivity toexogenous insulin in insulin-resistant HepG2 cells

Following 10−9 M insulin incubation, of HepG2 cell therewas a significant decrease in the consumption of extracellularglucose in controls with 10−7 M insulin pretreatment (Pb0.01)compared with controls without insulin pretreatment (Table 3).Insulin at the final concentration of 10−9 M significantly in-creased this lowered consumption of extracellular glucose inHepG2 cells pretreated with 10−7 M insulin combined withmetformin (10−5 g/ml) or AC (10−5, 10−6, 10−7 g/ml), re-spectively. Intracellular glycogen was decreased in AC (10−5

and 10−6 g/ml)-treated HepG2 cells. No change was noted inmetformin-treated HepG2 cells. The effect of AC at 10−5 g/mlwas comparable to that of metformin at 10−5 g/ml.

Table 3Effect of the ethanolic extracts of Ananas comosus L. leaves (AC) on sensitivityto exogenous insulin in insulin-resistant HepG2 cells pretreated with 10−7 Minsulin

Groups Dosage(g/ml)

Consumption ofextracellular glucose(nmol/mg cell protein)

Intracellular glycogen(μg/mg cell protein)

Blank control 49.08±1.73 0.190±0.012Control 46.79±1.53## 0.204±0.018Metformin 10−5 50.75±1.07⁎⁎ 0.214±0.016AC 10−4 47.44±0.55 0.185±0.023

10−5 49.77±0.83⁎⁎ 0.166±0.010⁎⁎

10−6 49.28±1.82⁎ 0.180±0.015⁎

10−7 48.34±0.26⁎ 0.188±0.010

Data were expressed as Mean±SD (n=5). ##Pb0.01 vs. blank control without10−7 M insulin pretreatment by ANOVA; ⁎Pb0.05, ⁎⁎Pb0.01 vs. control with10−7 M insulin pretreatment by ANOVA followed by Newman–Keuls test.

5. Discussion

STZ or alloxan induced rats produced hyperglycemia. Bothcompounds are widely used to mimic diabetic patients and toevaluate hypoglycemic or related effects of compounds andextracts (Kordowiak et al., 2000; Pari and Saravanan, 2002).Single administration of AC could enhance exogenous insulinsensitivity in diabetic rats. However, we did not know if AC had adirect hypoglycemic effect in the diabetic rats. Interestingly, nosignificant changewas observed in the diabetic rats whenACwasseparately used (without the injection of exogenous insulin) in ratsin an additional trial (data not shown). Therefore, this effect of ACmight be associated with the promotion of insulin sensitivity, andnot a result from a direct hypoglycemic effect of AC. Metformin,believed to alleviate insulin resistance in the presence of insulin(Bailey, 1993), was selected as positive control to evaluateeffectiveness of themodel.Metformin has been shown to improvethe insulin sensitivity in type 2 diabetes by activating post-recep-tor insulin signaling pathways. AC at a dose of 0.4 g/kg was moreeffective thanmetformin (0.32 g/kg). To challengewith exogenousinsulin is an acute test used to evaluate effect of AC on improvinginsulin sensitivity and to determine the most effective dose in thistrial. In the other sub-acute trials, we have adopted this mosteffective dose (0.4 g/kg). However, whether AC exerted the effectby the action mechanism of metformin remained undetermined.

Insulin resistance was associated with a loss of tolbutamide-induced blood glucose lowering action (Chi et al., 1998; Changet al., 1999). Tolbutamide belongs to hypoglycemic sulphony-lureas to stimulate insulin release in diabetic patients. If patientsshow insulin resistance, they will show a loss of tolbutamide-induced blood glucose lowering action. However, insulin sen-sitizer, thiazolidinediones, may strengthen the hypoglycaemiceffect of tolbutamide. Tolbutamide had no significant effect indiabetic rats in the present trial. It indicates these diabetic ratsmight be associated with insulin resistance. Our results showedthat AC enhanced tolbutamide-induced blood glucose loweringaction, and then inhibited the development of insulin resistancein insulin resistant rats induced by low-dose STZ and high-fatdiets. These results suggest that AC can enhance insulin sen-sitivity. In addition, if AC could stimulate insulin secretion, itshould have a hypoglycaemic effect in normal rats because theirinsulin release is in good condition, e.g., tolbutamide had ahypoglycaemic effect in normal rats. Interestingly, no signifi-cant effect was observed in normal rats when AC was separatelyused without the administration of tolbutamide, further sug-gesting the idea that AC affects insulin sensitivity, and does notplay a direct hypoglycaemic role. It seems that AC exerts ahypoglycaemic effect by enhancing insulin sensitivity not bystimulating insulin secretion. However, the precise mechanismsof AC need the further investigation.

Following intravenous insulin and glucose infusion test,insulin resistance was also produced in low-dose alloxan-treatedand high-fat diet fed rats, whichmay become a kind of new animalmodel to simulate type 2 diabetes. Here, the reason why we usedalloxan instead of STZ was because not only alloxan was cheaperthan STZ but also it worked as well as STZ in the preliminarystudy. 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 improvesexogenous insulin sensitivity. Following glucose infusion, asmaller increase in blood glucose was observed in diabetic rats,suggesting that AC enhances the insulin function in glucosedisposal in diabetic rats. The increase in glucose disposal of ACmay be not related to stimulate insulin secretion because: AC hadno effect in normal rats; endogenous insulin was much less thanexogenous insulin and was neglectable in diabetic rats. ACameliorated insulin sensitivity in this model, a result that is con-sistent with the observations above. Fenofibrate, a lipid-loweringdrug, also improved insulin sensitivity, which indicated that in-sulin resistance in this model might develop from a high-fat dietfed. In addition, some studies reported that fenofibrate has a goodhypoglycaemic effect in type 2 diabetes (Damci et al., 2003).Therefore, fenofibrate was selected as a suitable positive control.However, whether AC improved insulin sensitivity was associ-ated with its hypolipidemic effect remained unknown.

Further, an insulin-resistant HepG2 cell model was developedafter 24 h of 10−7 M insulin incubation. Here, consumption ofextracellular glucose content in hepatic cells was adopted toevaluate the disposal of them to extracellular glucose. The liverplays an important role in blood glucose control. Insulin resis-tance in hepatic cells results in the decease in extrahepatic glucosedisposal. High-concentration insulin induced insulin resistance inHepG2 cells and showed an insensitive response to low-con-centration insulin. AC significantly improved insulin action andinhibited the development of insulin resistance in HepG2 cells,which was consistent with the results in rats. Metformin alsoimproved insulin action. AC-treated HepG2 cells contain a lowerlevel of intracellular glycogen than controls, suggesting that ACactions differ from those for metformin.

Together, AC improved insulin sensitivity in animals of insulinresistance, which might be associated with enhancement of in-sulin action in hepatic cells. However, more precise mechanismsrequire further investigation. Natural products often have morepotential benefits than synthetic compounds in treatment of dia-betes and its complications (Xie et al., 2005b). An agent thatimproves insulin sensitivity, named insulin sensitizer, has recentlybeen developed (Scheen and Lefebvre, 1999). However, the cli-nical application remains limited due to its toxic effect (Forman etal., 2000). AC appears to be of little toxicity in our chronic–toxicexperiments of rats and Beagle dogs (data not shown). Apart fromthe above, AC has hypolipidemic, hypoglycemic and anti-ox-idative activities in diabetic rats (Xie et al., 2005a). Further,cardiovascular protection effects of AC have been determined inour laboratory (data not shown). Therefore, AC seems promisingto type 2 diabetic patients as an alternativemedication since type 2diabetic patients are associated with insulin resistance, hypergly-cemia, hyperlipidemia, and cardiovascular complications (Her-mayer, 2004; Rett, 1999).

In conclusion, AC could improve insulin sensitivity in type 2diabetic rats, which might be associated with enhancement ofinsulin action in hepatic cells. Thus, AC is hopeful to develop to anadjuvant for handling of diabetic patients with clinically ma-nifested insulin resistance in the coming future. Further, phar-macological and phytochemical investigations are underway toelucidate in detail the molecule mechanism of the effects of A.

comosus leaves on diabetic rats and to determine its activecompounds.

Acknowledgments

The study was supported by the Science and DevelopmentFoundation Tsinghua University (No. A2005568).

References

Bailey, C.J., 1993. Metformin—an update. Gen. Pharmacol. 24, 1299–1309.Chang, S.L., Lin, J.G., Chi, T.C., Liu, I.M., Cheng, J.T., 1999. An insulin-

dependent hypoglycaemia induced by electroacupuncture at the Zhongwan(CV12) acupoint in diabetic rats. Diabetologia 42, 250–255.

Chen, D., Wang, M.W., 2005. Development and application of rodent modelsfor type 2 diabetes. Diabetes Obes. Metab. 7, 307–317.

Chi, J.T., Liu, I.M., Cheng, J.T., 1998. A simple and rapid model of insulin-resistance in Wistar rats. Chin. Pharm. J. 50, 113–121.

Cordain, L., Eades, M.R., Eades, M.D., 2003. Hyperinsulinemic diseases ofcivilization: more than just Syndrome X. Comp. Biochem. Physiol., A 136,95–112.

Damci, T., Tatliagac, S., Osar, Z., Ilkova, H., 2003. Fenofibrate treatment isassociated with better glycemic control and lower serum leptin and insulinlevels in type 2 diabetic patients with hypertriglyceridemia. Eur. J. Intern.Med. 14, 357–360.

Egger, M., Davey, S.G., Stettler, C., Diem, P., 1997. Risk of adverse effects ofintensified treatment in insulin-dependent diabetes mellitus: a meta-analysis.Diabet. Med. 14, 919–928.

Fanger, B.O., 1987. Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents. Anal.Biochem. 162, 11–17.

Forman, L.M., Simmons, D.A., Diamond, R.H., 2000. Ann. Intern. Med. 132,118–121.

Gomez-Lechon, M.J., Ponsoda, X., Castell, J.V., 1996. A microassay formeasuring glycogen in 96-well-cultured cells. Anal. Biochem. 236, 296–301.

Hermayer, K.L., 2004. Treatment of lipids and type 2 diabetes. Curr. Cardiol.Rep. 6, 443–450.

Kopelman, P., 2004. The metabolic syndrome as a clinical problem. Nestle Nutr.Workshop Ser. Clin. Perform. Progr. 9, 77–88 (discussion 88-92).

Kordowiak, A.M., Dudek, B., Grybos, R., 2000. Influence of sodium bis(oxalato)oxovanadate(IV) on phospholipids in liver Golgi fractions fromcontrol and streptozotocin-diabetic rats. Comp. Biochem. Physiol., C 125,11–16.

Li, C.G., Ning, G., Chen, J.L., 1999. Establishing and identifying insulin-resistant HepG2 cell line. Chin. Diabetes 7, 198–200 (in Chinese).

Mensah, G.A., Mokdad, A.H., Ford, E., Narayan, K.M., Giles, W.H., Vinicor, F.,Deedwania, P.C., 2004. Obesity, metabolic syndrome, and type 2 diabetes:emerging epidemics and their cardiovascular implications. Cardiol. Clin. 22,485–504.

Milani, E., Nikfar, S., Khorasani, R., Zamani, M.J., Abdollahi, M., 2005.Reduction of diabetes-induced oxidative stress by phosphodiesterase in-hibitors in rats. Comp. Biochem. Physiol., C 140, 251–255.

Reed, M.J., Meszaros, K., Entes, L.J., Claypool, M.D., Pinkett, J.G., Gadbois,T.M., Reaven, G.M., 2000. A new rat model of type 2 diabetes: the fat-fed,streptozotocin-treated rat. Metabolism 49, 1390–1394.

Rett, K., 1999. The relation between insulin resistance and cardiovascularcomplications of the insulin resistance syndrome. Diabetes Obes. Metab. 1,S8–S16.

Pari, L., Saravanan, G., 2002. Antidiabetic effect of Cogent db, a herbal drug inalloxan-induced diabetes mellitus. Comp. Biochem. Physiol., C 131, 19–25.

Scheen, A.J., 2004. Management of the metabolic syndrome. MinervaEndocrinol. 29 (2), 31–45.

Scheen, A.J., Lefebvre, P.J., 1999. Troglitazone: andtihyperglycemic activity andpotential role in the treatment of type 2 diabetes. Diabetes Care 22, 1568–1577.

Song, L.L., 1999. Chinese Herbs. Administrant Department of National ChineseTraditional Medicine, Shang Hai, chapter 8, pp. 296–297 (in Chinese).

435W. Xie et al. / Comparative Biochemistry and Physiology, Part C 143 (2006) 429–435

Sripanidkulchai, B., Na-nakorn, S., Wongpanich, V., Tanyakupta, P., 2000. Abehavioral investigation on utilization of medicinal plants for dysuria atPon District, Khon Kaen. Khon Kaen University Research Journal, vol. 5,pp. 4–10.

Sripanidkulchai, B., Wongpanich, V., Laupattarakasem, P., Suwansaksri, J.,Jirakulsomchok, D., 2001. Diuretic effects of selected Thai indigenousmedicinal plants in rats. J. Ethnopharmacol. 75, 185–190.

Trevaskis, J., McMillan, J.S., Windmill, K., Walder, K., Collier, G.R., 2004.Identification of hypothalamic genes implicated in the development ofobesity in Psammomys obesus using differential display PCR. Comp.Biochem. Physiol., B 137, 65–73.

Wu, Y.C., Hsu, J.H., Liu, I., Liou, S.S., Su, H.C., Cheng, J.T., 2002. Increase ofinsulin sensitivity in diabetic rats received Die-Huang-Wan, a herbal mixtureused in Chinese traditional medicine. Acta Pharmacol. Sin. 23 (12),1181–1187.

Xie, W.D., Du, L.J., 2005. High-cholesterol diets impair short-term retention ofmemory in alloxan-induced diabetic mice, but not acquisition of memory inprediabetic mice. Life Sci. 77, 481–495.

Xie, W.D., Xing, D.M., Sun, H., Wang, W., Ding, Y., Du, L.J., 2005a. Theeffects of Ananas comosus L. leaves on diabetic–dyslipidemic rats inducedby alloxan and a high-fat/high-cholesterol diet. Am. J. Chin. Med. 33,95–105.

Xie, W.D., Xing, D.M., Zhao, Y.N., Su, H., Meng, Z., Chen, Y.Y., Du, L.J.,2005b. A new tactic to treat postprandial hyperlipidemia in diabetic rats withgastroparesis by improving gastrointestinal transit. Eur. J. Pharmacol. 510,113–120.

Xu, J.S., Ma, M.W., Purcell, W.M., 2003. Characterisation of some cytotoxicendpoints using rat liver and HepG2 spheroids as in vitro models and theirapplication in hepatotoxicity studies. I. Glucose metabolism and enzymerelease as cytotoxic markers. Toxicol. Appl. Pharmacol. 189, 100–111.

Zhang, F., Ye, C., Li, G., Ding,W., Zhou, W., Zhu, H., Chen, G., Luo, T., Guang,M., Liu, Y., Zhang, D., Zheng, S., Yang, J., Gu, Y., Xie, X., Luo, M., 2003.The rat model of type 2 diabetic mellitus and its glycometabolism characters.Exp. Anim. 52, 401–407.