Antihyperglycemic Effect of Andrographolide in Streptozotocin-Induced Diabetic Rats

• Abstract
• Introduction
• Materials and Methods
• Animal models
• Effect of andrographolide on plasma glucose
• Intravenous glucose challenge test
• Determination of plasma glucose
• Measurement of glucose uptake into soleus muscle
• Effect on gene expression
• Data analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

The antihyperglycemic action of andrographolide, an active principle in the leaves of Andrographis paniculata (Burm. f.) Nees, was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral treatment of andrographolide decreased the plasma glucose concentrations of STZ-diabetic rats in a dose-dependent manner. Similar treatment with andrographolide also decreased the plasma glucose in normal rats and the maximal effect was more marked than that in STZ-diabetic rats. Andrographolide at the effective dose (1.5 mg/kg) significantly attenuated the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. In the isolated soleus muscle of STZ-diabetic rats, andrographolide enhanced the uptake of radioactive glucose in a concentration-dependent manner. Moreover, the mRNA and protein levels of the subtype 4 form of the glucose transporter (GLUT4) in soleus muscle were increased after repeated intravenous administration of andrographolide in STZ-diabetic rats for 3 days. These results suggest that andrographolide can increase the glucose utilization to lower plasma glucose in diabetic rats lacking insulin.

Introduction

Insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) rank highly among the top ten causes of mortality throughout the world. Diabetes often leads to disability from the vascular complications of coronary artery disease and cerebrovascular disease, renal failure, blindness, and limb amputation in addition to neurological complications and premature death [1]. Treatment of DM by insulin and/or oral drugs fails to prevent these complications in many patients indicating that the additional treatment will be helpful. Andrographis paniculata may be the new therapeutic agent for diabetes. In oriental medicine, Andrographis paniculata was emphasized for its anti-oxidation effect, hepatoprotective effect and anti-inflammatory effect [2], [3]. Also, it has been documented that ethanol extracts of Andrographis paniculata decreased the plasma glucose in normal rats and STZ-diabetic rats [4]. Andrographolide is mentioned as the principle with highest content in Andrographis paniculata [5], [6], [7]. Recently, the anti-inflammatory mechanism of andrographolide was investigated [7]. However, andrographolide has been documented to show no effect on blood sugar [8]. Thus, we used andrographolide (Fig. [1]) to characterize the effect on glucose metabolism in diabetic rats.

Materials and Methods

Animal models

Male Wistar rats, aged 8 - 10 weeks (200 - 250 g body weight), were obtained from the Animal Center of National Cheng Kung University Medical College. Diabetic rats were prepared by giving an intravenous injection of STZ (Sigma-Aldrich Chemical Co., St. Louis, MO) at 65 mg/kg to the fasting rats. Rats with plasma glucose concentrations of 20 mmol/L or greater in addition to polyuria and other diabetic features were considered as having IDDM. Also, the plasma insulin level in STZ-diabetic rats decreased to 1.3 ± 0.7 pmol/L (n = 8), markedly lower than the level for the normal rats (165.1 ± 2.7 pmol/L; n = 8) showing IDDM. All studies were carried out 2 weeks after the injection of STZ. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act.

Effect of andrographolide on plasma glucose

Andrographolide was purchased commercially from Sigma-Aldrich Chemical Co. (St. Louis, MO) with a purity higher than 98 % and the solution was prepared by dissolving the substance in ethanol for dilution with normal saline to the desired dose or concentration. A solution prepared in same way without the addition of andrographolide was taken as vehicle. The fasting STZ-diabetic rats received an oral treatment of andrographolide at the desired doses and blood samples (0.1 mL) were collected under sodium pentobarbital anesthesia (30.0 mg/kg, i. p.) from the tail vein for measurement of plasma glucose. In the preliminary experiments, andrographolide at 1.5 mg/kg was found to produce the maximal plasma glucose lowering effect in STZ-diabetic rats 60 min after oral treatment. Thus, the effect of andrographolide on plasma glucose was determined using blood samples collected after 60 min. Control rats received a similar treatment of vehicle at the same volume. Also, the response to oral treatment of metformin (Sigma-Aldrich Chemical Co, St. Louis, MO) or intravenous injection of bovine insulin (Novo Industrias, Bagsvaerd, Denmark) in STZ-diabetics rats was taken as the positive control [9].

Intravenous glucose challenge test

An intravenous glucose challenge test (IVGCT) was performed according to the method described previously [10]. Briefly, the basal plasma glucose concentration was obtained from samples from the tail vein of Wistar rats under anesthesia with sodium pentobarbital (30.0 mg/kg, i. p.) before the IVGCT. A solution of andrographolide at 1.5 mg/kg or the same volume of vehicle was given to rats by oral treatment. At 30 min later, blood samples (0.1 mL) from the tail vein were drawn and indicated as 0 min. Then, a glucose dose of 1 g/kg was injected through the femoral vein of the rats. Rats receiving a similar injection of saline at the same volume were taken as control. Blood samples (0.1 mL) from the tail vein were drawn at 5, 10, 20, 30, 60, 90 and 120 min following the glucose injection for the measurement of the plasma glucose concentrations. Rats were maintained under anesthesia by pentobarbital throughout the procedure.

Determination of plasma glucose

Blood samples (0.1 mL) were collected in a chilled syringe containing 10 IU heparin from the tail vein of rats under anesthesia with sodium pentobarbital (30.0 mg/kg, i. p.). Blood samples were then centrifuged at 13,000 rpm for 3 min and an aliquot (15 μL) of plasma was added to 1.5 mL of 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. The concentration of plasma glucose was then estimated via an analyzer (Quik-Lab, Ames, Miles Inc., Elkhart, Indiana 46 515, U.S.A.) with samples run in duplicate as described previously [11].

Measurement of glucose uptake into soleus muscle

Glucose uptake was determined using the uptake of the radioactive glucose analogue, 2-[1 - ¹⁴ C]-deoxy-D-glucose (2-DG) (New England Nuclear, Boston, U.S.A.), as described previously [11]. The obtained soleus muscles were placed in 3 mL of Krebs-Ringer bicarbonate buffer (KRBB) (37 °C, pH 7.4) containing 1 mmol/L glucose, 1 % fatty acid-free bovine serum albumin (BSA) under aeration with 5 % CO₂ in O₂. After pre-incubation for 30 min, the muscle tissue was incubated with 1.0 nmol/L bovine insulin or andrographolide at the desired concentrations for 30 min and then with 50 μL KRBB containing 2-DG (1 μCi/mL) for 5 min at 37 °C in the shaking water bath under aeration. Reactions were terminated by quickly blotting the muscles and dissolving them in 0.5 mL of 0.5 N NaOH for 45 min before neutralization with 0.5 mL of 0.5 N HCl. After centrifugation, 800 μL of each supernatant were mixed with 1 mL of aqueous counting scintillant and the radioactivity was determined using a β-counter (Beckman LS6000). Non-specific uptake of 2-DG, assessed after an incubation with 20 μmol/L cytochalasin B (Sigma-Aldrich Chemical Co., St. Louis, MO) to block transportation, was subtracted from the total muscle-associated radioactivity. Specific 2-DG uptake was then expressed as the percentage of basal uptake taken from soleus muscle incubating with KRBB only.

Effect on gene expression

Both STZ-diabetic rats and normal rats received the oral treatment of andrographolide at an effective dose (1.5 mg/kg) every 8 h, three times daily (t. i. d.). In the preliminary experiments, andrographolide (1.5 mg/kg) was found to significantly modify the mRNA and protein levels for GLUT4 after 3 days treatment. Thus, the effect of andrographolide on the mRNA and protein levels for GLUT4 was determined using samples collected after 3 days treatment. After the final treatment, the fasting animals were sacrificed. Soleus muscle was immediately removed, frozen in liquid nitrogen and stored at -70 °C for Northern and Western blot analyses that were carried out as our previous method [12]. Blood samples were also collected from the tail vein of these rats before sacrifice.

Data analysis

Results of plasma glucose lowering activity were calculated as percentage decrease of the initial value according to the formula: (Gi - Gt)/Gi × 100 % where Gi was the initial glucose concentration and Gt was the plasma glucose concentration after treatment with andrographolide or another testing agents. Data are expressed as the mean ± SEM for the number (n) of animals in the group as indicated in tables and figures. Repeated measures analysis of variance (ANOVA) was used to analyze the changes in plasma glucose and other parameters. Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. P < 0.05 was considered statistically significant.

Results and Discussion

In the present study, we found that the oral treatment of andrographolide could lower plasma glucose effectively in STZ-diabetic rats. As shown in Fig. [2], a dose-dependent reduction of plasma glucose was observed in STZ-diabetic rats after oral treatment with andrographolide at the dose from 0.5 to 1.5 mg/kg. Also, we employed insulin as positive control. An intravenous injection of insulin at 1.0 IU/kg reduced the plasma glucose of STZ-diabetic rats to 15.0 ± 0.7 mmol/L which was markedly lower than the basal level (26.6 ± 1.1 mmol/L). The maximal plasma glucose lowering activity of andrographolide in STZ-diabetic rats was 23.7 ± 3.5 % at 1.5 mg/kg. Thus, 1.5 mg/kg of andrographolide was employed in subsequent experiments. Moreover, the plasma glucose lowering activity of andrographolide in normal rats was more marked than that in STZ-diabetic rats at higher doses (Fig. [3]). The plasma glucose lowering activities of metformin (100 mg/kg) as positive control in normal rats and STZ rats are 26.3 ± 3.5 % and 19.8 ± 2.9 %, respectively. The effect of andrographolide is thus similar to that of metformin. Different to the previous report [8], our results are consistent with the data in a recent paper [4] because andrographolide is dissolved in ethanol for investigation in the present study.

On the basis that the intravenous glucose challenge test (IVGCT) is used to characterize the ability of rats to utilize glucose from the circulation, the IVGCT was then carried out. As shown in Fig. [4], the basal plasma glucose concentration in Wistar rats was 5.2 ± 0.2 mmol/L. Sixty minutes after treatment, the plasma glucose concentration was decreased to 6.4 ± 0.3 mmol/L in rats receiving oral treatment with andrographolide (1.5 mg/kg). However, the plasma glucose concentration was 5.1 ± 0.3 mmol/L in vehicle-treated rats, which was not different from the basal plasma glucose level of rats. Five minutes after glucose injection, the plasma glucose concentration was elevated to 16.5 ± 0.2 mmol/L in vehicle-treated rats and was 14.7 ± 0.4 mmol/L in andrographolide-treated rats (Fig. [4]). The increase of plasma glucose produced by the glucose injection was significantly reduced in the rats pretreated with andrographolide. The plasma glucose in rats pretreated with andrographolide remained significantly lower 30 min after glucose injection compared to the vehicle-treated group (Fig. [4]). No statistical difference (P > 0.05) was obtained for the plasma glucose concentration in rats receiving glucose injection at 90 min later between the andrographolide-treated group and vehicle-treated controls.

Skeletal muscle is a major site of glucose disposal [13]. Glucose transport, which depends on insulin-stimulated translocation of glucose carriers to the cell membrane, is the rate-limiting step in the carbohydrate metabolism of skeletal muscle [14]. Insulin-stimulated glucose utilization is the major site for regulation of plasma glucose concentrations [15]. IDDM is an abnormal metabolic state characterized by an insulin defect involving muscle and other tissues. Thus, the effect on glucose uptake by andrographolide was investigated to characterize the direct influence on glucose utilization. As the positive control, specific glucose uptake (2-DG uptake) into the soleus muscle of STZ-diabetic rats was increased to about 212.7 ± 6.5 % by incubation with 1.0 nmol/L bovine insulin taking the basal level of glucose uptake as 100 %. A similar incubation with andrographolide for 30 min increased the specific glucose uptake into isolated soleus muscle of STZ-diabetic rats in a concentration-dependent manner from 0.01 to 1.0 μmol/L (Fig. [5]). However, the maximal activity of andrographolide was still smaller (P < 0.05) than that of bovine insulin (1.0 nmol/L), the positive control. These results suggest that andrographolide can increase the utilization of glucose in peripheral tissue via an insulin-independent mechanism. Otherwise, the effective concentration of andrographolide herein is near to that produced an anti-inflammatory action [7]. Further studies are needed to elucidate the action mechanisms of andrographolide for glucose homeostasis and the relation with anti-inflammation in detail.

The glucose transporters (GLUT) mediate glucose transport across the cell membrane. The glucose transporters subtype 4 (GLUT4) is predominant in skeletal muscle [16]. A reduction in insulin-mediated glucose uptake caused by decreasing expression of GLUT4 mRNA and protein in diabetes has been observed [17], [18]. It is possible that andrographolide can enhance the glucose uptake via an effect on gene expression of GLUT4. Fig. [6] shows the representative responses of mRNA level for GLUT4 in isolated soleus muscle from the vehicle- or andrographolide-treated STZ-diabetic rats as determined by Northern blot analysis using β-actin as the internal standard. The mRNA level of GLUT4 in isolated soleus muscle of the vehicle-treated STZ-diabetic rats was only about 51 % of that from the vehicle-treated normal rats. It has been documented that long-term exposure is required for the activation of mRNA level in cells [19]. Repeated treatment of STZ-diabetic rats with andrographolide (1.5 mg/kg) three times daily (t. i. d.) for 3 days resulted in a marked elevation of the GLUT4 mRNA level in isolated soleus muscle to a level near that of vehicle-treated normal rats (Fig. [6] A). The quantification of the mRNA levels for changes of GLUT4 is also shown in Table [1]. Plasma glucose concentrations of STZ-diabetic rats were significantly (P < 0.01) decreased to 16.5 ± 3.1 mmol/L after repeated treatment with andrographolide (1.5 mg/kg, t. i. d.) for 3 days, as compared to vehicle-treated STZ-diabetic rats (26.1 ± 3.3 mmol/L). However, the plasma glucose concentration in these andrographolide-treated rats was still higher than that in the vehicle-treated normal rats (5.4 ± 0.6 mmol/L, P < 0.01).

A similar action of andrographolide was found on the protein level of GLUT4 in isolated soleus muscle determined by Western blot (Fig. [6] B). The protein level of GLUT4 in the soleus muscle of vehicle-treated STZ-diabetic rats was only about 40 % that of vehicle-treated normal rats (Fig. [6] B). After andrographolide (1.5 mg/kg, t. i. d.) treatment for 3 days, the GLUT4 protein level was elevated to about 80 % of the level of vehicle-treated normal rats (Table [1]). From the raise in both protein and mRNA levels of GLUT4 by andrographolide, an increase of GLUT4 gene expression can be considered as one of the actions of mechanism of andrographolide. Treatment with metformin as a positive control showed a similar change of GLUT4 gene expression (Table [1]).

In conclusion, the data obtained suggest that oral treatment of andrographolide can lower plasma glucose in STZ-diabetic rats through an increase of glucose utilization. Thus, andrographolide may develop as an attractive adjuvant for the handling of diabetic disorders in the future.

Acknowledgements

We are grateful to Professor C. Makepeace and Professor S. S. Liu for kindly providing us with the plasmid containing cDNA. Thanks are also due to Professor Y.C. Tong for editing the manuscript.

References

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Professor Juei-Tang Cheng

Department of Pharmacology

College of Medicine

National Cheng Kung University

Tainan City

Taiwan 70101

R.O.C.

Phone: +886-6-237-2706

Fax: +886-6-238-6548

Email: jtcheng@mail.ncku.edu.tw

References

• 1 Weidmann P, de Courten M, Boehlen L M. Pathogenesis and treatment of hypertension associated with diabetes mellitus.  Am Heart J. 1993;  125 1498-513
  CrossrefPubMedSearch in Google Scholar
• 2 Trivedi N P, Rawal U M. Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice.  Indian J Exp Biol. 2001;  39 41-6
  PubMedSearch in Google Scholar
• 3 Panossian A, Hovhannisyan A, Mamikonyan G, Abrahamian H, Hambardzumyan E, Gabrielian E, Goukasova G, Wikman G, Wagner H. Pharmacokinetic and oral bioavailability of andrographolide from Andrographis paniculata fixed combination Kan Jang in rats and human.  Phytomedicine. 2000;  7 351-64
  CrossrefPubMedSearch in Google Scholar
• 4 Zhang X F, Tan B K. Antihyperglycaemic and anti-oxidant properties of Andrographis paniculata in normal and diabetic rats.  Clin Exp Pharmacol Physiol. 2000;  27 358-63
  CrossrefPubMedSearch in Google Scholar
• 5 Lu X L, Zhang S L, Wang Z S. Analysis of andrographolide compounds. I. Ion pair high performance liquid chromatographic analysis of andrographolide derivatives.  Yao Xue Xue Bao. 1981;  16 182-9
  PubMedSearch in Google Scholar
• 6 Maiti P C. Andrographolide: The active principle of Kalmegh.  Bull Bot Surv India. 1964;  6 63-5
  PubMedSearch in Google Scholar
• 7 Shen Y C, Chen C F, Chiou W F. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect.  Br J Pharmacol. 2002;  135 399-406
  CrossrefPubMedSearch in Google Scholar
• 8 Ahmed M, Talukder S A. Studies on the hypoglycemic activity of Kalmegh (Andrographis paniculata Nees.) on the blood sugar level of rats.  Bangladesh Pharm J. 1977;  6 21-4
  PubMedSearch in Google Scholar
• 9 Cheng J T, Liu I M, Chi T C, Su H C, Chang C G. Metformin-like effects of Quei Fu Di Huang Wan, a Chinese herbal mixture, on streptozotocin-induced diabetic rat.  Horm Metab Res. 2001;  33 727-32
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Liu I M, Chi T C, Hsu FL,Chen C F, Cheng J T. Isoferulic acid as active principle from the rhizoma of Cimicifuga dahurica to lower plasma glucose in diabetic rats.  Planta Med. 1999;  65 712-4
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Liu I M, Hsu F L, Chen C F, Cheng J T. Antihyperglycemic action of isoferulic acid in streptozotocin-induced diabetic rats.  Br J Pharmacol. 2000;  129 631-6
  CrossrefPubMedSearch in Google Scholar
• 12 Cheng J T, Liu I M, Chi T C, Tzeng T F, Lu F H, Chang C J. Plasma glucose-lowering effect of tramadol in streptozotocin-induced diabetic rats.  Diabetes. 2001;  50 2815-21
  PubMedSearch in Google Scholar
• 13 Baron A D, Brechtel G, Wallace P, Edelman S V. Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans.  Am J Physiol. 1988;  255 E769-74
  PubMedSearch in Google Scholar
• 14 Ziel F H, Venkatesan N, Davidson M B. Glucose transport is rate limiting for skeletal muscle glucose metabolism in normal and STZ-induced diabetic rats.  Diabetes. 1988;  37 885-90
  PubMedSearch in Google Scholar
• 15 Hollenbeck C, Reaven G M. Variations in insulin-stimulated glucose uptake in healthy individuals with normal glucose tolerance.  J Clin Endocrinol Metab. 1987;  64 1169-73
  PubMedSearch in Google Scholar
• 16 Pessin J E, Bell G I. Mammalian facilitative glucose transporter family: structure and molecular regulation.  Annu Rev Physiol. 1992;  54 911-30
  CrossrefPubMedSearch in Google Scholar
• 17 Berger J, Biswas C, Vicario P P, Strout H V, Saperstein R, Pilch P F. Decreased expression of the insulin-responsive glucose transporter in diabetes and fasting.  Nature. 1989;  340 70-2
  CrossrefPubMedSearch in Google Scholar
• 18 Sivitz W I, Desautel S L, Kayano T, Bell G I, Pessin J E. Regulation of glucose transporter messenger RNA in insulin-deficient states.  Nature. 1989;  340 72-4
  CrossrefPubMedSearch in Google Scholar
• 19 Henriksen E J, Bourey R E, Rodnick K J, KoranyI L, Permutt M A, Holloszy J O. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles.  Am J Physiol. 1990;  259 E593-8
  PubMedSearch in Google Scholar

Professor Juei-Tang Cheng

Department of Pharmacology

College of Medicine

National Cheng Kung University

Tainan City

Taiwan 70101

R.O.C.

Phone: +886-6-237-2706

Fax: +886-6-238-6548

Email: jtcheng@mail.ncku.edu.tw