Mediation of β-Endorphin by Ginsenoside Rh2 to Lower Plasma Glucose in Streptozotocin-Induced Diabetic Rats

• Abstract
• Introduction
• Materials and Methods
• Animal models
• Effect of Rh2 on plasma glucose or β-endorphin in STZ-diabetic rats
• Effect of Rh2 on plasma glucose in opioid μ-receptor knockout diabetic mice
• Effect of Rh2 on gene expression of glucose transporter subtype 4 (GLUT 4)
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

We investigated the plasma glucose-lowering mechanism(s) of Rh2, a ginsenoside derived from Panax ginseng, in rats with streptozotocin-induced diabetes (STZ-diabetic rats). After intravenous injection over 120 min into fasting STZ-diabetic rats, Rh2 decreased plasma glucose in a dose-dependent manner. In parallel to the lowering of plasma glucose, an increase of plasma β-endorphin-like immunoreactivity was observed. In addition, naloxone and naloxonazine at doses sufficient to block opioid μ-receptors inhibited the plasma glucose-lowering action of Rh2 in genetically wild-type, diabetic mice. In contrast, Rh2 failed to lower plasma glucose in opioid μ-receptor knockout diabetic mice. An increase in gene expression at both the mRNA and protein levels of glucose transporter subtype 4 (GLUT 4) was observed in soleus muscle obtained from STZ-diabetic rats treated with Rh2 three times daily for one day; this increase in expression was absent when opioid μ-receptors were blocked. In conclusion, our results suggest that ginsenoside Rh2 may lower plasma glucose in STZ-diabetic rats based on an increase in β-endorphin secretion that activates opioid μ-receptors thereby resulting in an increased expression of GLUT 4.

Introduction

Diabetes is one of the most serious chronic disorders, and it often leads to disability resulting from vascular complications of coronary artery disease, cerebrovascular disease, renal failure, blindness, limb amputation, and neurological complications, as well as premature death [1]. In order to bring the plasma glucose level as close to the normal range as possible, dietary restrictions, exercise, and oral glucose-lowering agents are widely applied in cases where insulin is not necessary. Currently, there is an enormous increase in the use of herbal and/or other alternative medicines for the treatment of diabetic morbidity.

The herbal remedies of ginseng have been used in Asia for several millennia. The most used species is Panax ginseng, or Asian or Korean red ginseng (P. ginseng, C.A. Meyer) [2]. In addition to modulation of psychological and immune functions, ginseng possesses neurotrophic and neuroprotective properties [3]. P. ginseng roots have been used to treat diabetes in Chinese traditional medicine. In animal studies of type 1 diabetes, the root of P. ginseng and other species including P. quinquefolius (American ginseng) showed antihyperglycemic activity [4], [5]. Recently, we observed that P. ginseng root can improve insulin resistance in rats receiving fructose-rich chow [6]. Thus, ginseng seems helpful in the prevention and/or management of diabetes. The major active components in P. ginseng are named ginsenosides, a series of derivatives of the triterpene dammarane. Ginsenoside Rh2 (Fig. [1]), an active component of P. ginseng root, has shown anti-tumor properties [7]. Thus, it is of interest to identify any effects of Rh2 on plasma glucose in type 1 diabetes.

Exogenous β-endorphin induces an increase of circulating insulin in normal and diabetic humans [8]. Activation of opioid μ-receptors by either exogenous β-endorphin or chemical agents may increase glucose utilization to lower plasma glucose in streptozotocin-induced diabetic rats (STZ-diabetic rats), the type 1 diabetes animal model [9], [10]. Therefore, we employed STZ-diabetic rats to investigate effects of Rh2 on plasma glucose in diabetic rats and to characterize the possible role of β-endorphin in this activity.

Materials and Methods

Animal models

Male Wistar rats aged 8 - 10 weeks (weight 200 - 250 g) were obtained from the Animal Center of the National Cheng Kung University Medical College. Male wild-type (BDF1 mice) and opioid μ-receptor knockout mice aged 8 - 10 weeks were obtained from Professor H. H. Loh (Department of Pharmacology, University of Minnesota Medical School. Minneapolis, MN, USA). STZ-diabetic rats were prepared by intravenous injection of STZ (60.0 mg/kg) (Sigma-Aldrich, Inc., Saint Louis, MO, USA) into the Wistar males. Mice with or without opioid μ-receptors received an intraperitoneal injection of STZ at 50.0 mg/kg to induce diabetes, according to previous reports [11]. Animals with plasma glucose concentrations of 20.0 mmol/L or greater in addition to polyuria and other diabetic features were considered to have type 1diabetes. Plasma insulin levels in STZ-diabetic rats decreased to 1.19 ± 0.62 pmol/L (n = 8). These levels are significantly lower than in normal rats (165.1 ± 6.2 pmol/L; n = 8) and document insulin deficiency. All studies were carried out two weeks after STZ injection. 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 Rh2 on plasma glucose or β-endorphin in STZ-diabetic rats

Granules of ginsenoside Rh2 (98 % pure) provided by Dr. Chen Wan-Chung (Department of Clinical Immunology, School of Medicine, Nihon University, Tokyo City, Japan) were dissolved in saline for injection. After fasting overnight, STZ-diabetic rats received intravenous injections of Rh2 at the desired doses, and blood samples (0.1 mL) were collected under sodium pentobarbital anesthesia (30.0 mg/kg, intraperitoneal [i. p.]) from the tail vein for measurement of plasma glucose concentrations or plasma β-endorphin-like immunoreactivity (BER). In preliminary experiments, Rh2 at 1.0 mg/kg produced the maximal plasma glucose-lowering effect in STZ-diabetic rats 120 min after a single intravenous injection. Thus, the effects of Rh2 on plasma glucose and BER were determined using blood samples collected 120 min after injection. STZ-diabetic rats that received the same volume of vehicle (distilled water with 0.9 % [w/v] NaCl) were used as controls. Further experiments were performed with pharmacological inhibitors (Research Biochemical Inc., Natick, MA, USA), such as the opioid μ-receptor antagonists naloxone and naloxonazine. These inhibitors were injected intravenously into fasting rats 30 min before injection of Rh2. Plasma glucose was measured with the glucose oxidase method using an analyzer (Quik-Lab, Ames, Miles Inc., Elkhart, IN, USA). Determination of BER was performed with an enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Penisula Lab. Inc., CA, USA).

Effect of Rh2 on plasma glucose in opioid μ-receptor knockout diabetic mice

Fasting STZ-diabetic mice with or without opioid μ-receptors received intravenous injections of Rh2 at 1.0 mg/kg, the dose that produced maximal plasma glucose lowering in STZ-diabetic rats. After 120 min, blood samples (0.1 mL) were collected from the lower eyelid under pentobarbital anesthesia (30.0 mg/kg, i. p.) using a chilled syringe containing 10 IU heparin.

Effect of Rh2 on gene expression of glucose transporter subtype 4 (GLUT 4)

STZ-diabetic rats received intravenous injections of vehicle or Rh2 (1.0 mg/kg) three times daily into the tail vein. Naloxonazine (1.5 mg/kg) was injected intravenously into another group of STZ-diabetic rats 30 min before injection of Rh2. In preliminary experiments, Rh2 significantly modified mRNA and protein levels of GLUT 4 in STZ-diabetic rats after one day of treatment. Thus, animals were sacrificed two days after treatment. Soleus muscle was immediately removed, frozen in liquid nitrogen, and stored at -70 °C for Northern or Western blot analysis as previously published [10]. Blood samples were also collected from the tail vein of fasting rats before sacrifice to evaluate changes of plasma glucose.

Statistical analysis

Plasma glucose-lowering activity was observed for animals that received Rh2. The results of the plasma glucose-lowering activity were calculated as percentage decrease from 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 injection of Rh2.

Data are expressed as the mean ± standard error (SE) for the number (n) of animals in the group. Repeated measures analysis of variance (ANOVA) was used to analyze changes in plasma glucose and other parameters. Dunnett range post-hoc comparisons were used to determine the source of significant differences, where appropriate. A P value < 0.05 was considered statistically significant.

Results and Discussion

Adequate control of blood glucose delays and/or prevents the progression of diabetic complications; this degree of control requires an intensive insulin regimen for patients with type 1 diabetes mellitus [12]. Actually, a decrease of plasma glucose is not dependent only on insulin. Evidence indicates that endogenous β-endorphin or opioid μ-receptor activation is a positive regulator in glucose homeostasis during an insulin-deficient state [9], [10]. In the present study, the plasma insulin level in STZ-diabetic rats was only about 1/120 of that of normal rats. This indicates that attenuation of endogenous insulin was negligible in this animal model. The plasma glucose-lowering action of the ginsenoside Rh2 was investigated with this animal model in order to clarify insulin-independent mechanism(s) focusing on the role of endogenous β-endorphin.

Oral medication is the most common route of administration of ginseng products; however, in order to rule out pharmacokinetic parameters, Rh2 was investigated in the present study with intravenous injection into test animals. Injection of Rh2 decreased plasma glucose in STZ-diabetic rats. The glucose level reached a plateau within 120 min that was maintained for 150 min or more (Fig. [2] A). The maximal plasma glucose-lowering effect in STZ-diabetic rats occurred at the dose of 1.0 mg/kg (Fig. [2] A). Increasing the Rh2 dose to 5.0 mg/kg had no additional effect. Similar to the lowering of plasma glucose, plasma BER gradually increased in the STZ-diabetic rats about 30 min after intravenous injection of Rh2 at 1.0 mg/kg. Maximal plasma BER levels were measured 120 min after injection (Fig. [2] B). A dose-dependent increase of the plasma BER level was also observed in the same rats that received Rh2 treatment. The effect proceeded in parallel to the lowering of plasma glucose (Fig. [2] B). An elevation of β-endorphin seems to be related to the plasma glucose-lowering action of Rh2 in the absence of insulin.

Endogenous β-endorphin has physiological actions that are mainly mediated by opioid μ-receptors [13]. Thus, blockers of opioid μ-receptors were employed to clarify the role of this receptor in the plasma glucose-lowering action of Rh2 in the STZ-diabetic rat model. The dose-dependent inhibitory effects of naloxone and naloxonazine on the action of Rh2 in STZ-diabetic rats are shown in Table [1]. In the presence of 1.5 mg/kg naloxone, plasma glucose in STZ-diabetic rats treated with 1.0 mg/kg Rh2 was reversed to a value near the basal level. Similar results were obtained with rats pretreated with 1.5 mg/kg naloxonazine. Moreover, both naloxone and naloxonazine failed to modify the basal plasma glucose level of STZ-diabetic rats even at the highest tested dose (Table [1]). Actually, neither naloxone nor naloxonazine modified Rh2-induced secretion of plasma BER (Table [1]). This suggests that the plasma glucose-lowering action of Rh2 in STZ-diabetic rats is mainly related to increased release of BER, which can activate opioid μ-receptors.

However, the possibility of multiple effects of these antagonists in addition to blockade of opioid μ-receptors should be a concern. Thus, we employed opioid μ-receptor knockout mice to induce type-1 like diabetes and used this model to confirm the role of opioid μ-receptors in the action of Rh2. Fig. [3] shows that plasma glucose concentrations in opioid μ-receptor knockout diabetic mice were not changed by Rh2 at a dose of 1.0 mg/kg. However, similar treatment with Rh2 (1.0 mg/kg) significantly (P < 0.05) decreased plasma glucose in diabetic mice with opioid μ-receptors. The plasma glucose-lowering level was decreased by 23.9± 2.4 % by Rh2 (1.0 mg/kg) in these wild-type diabetic mice and was similar to what has been seen in STZ-diabetic rats. These data support the essential role of opioid μ-receptors in the plasma glucose-lowering action of Rh2 in animals in an insulin-deficient state. Therefore, the possible mechanism through which Rh2 lowers plasma glucose in STZ-diabetic rats is mediated by increased secretion of β-endorphin that activates opioid μ-receptors.

In diabetes, elevation of blood glucose is related to reduced peripheral glucose utilization. Reduction in insulin-mediated glucose uptake caused by decreasing gene expression of the subtype-4 form of the glucose transporter (GLUT 4) has been reported in diabetic skeletal muscle, a major site for glucose disposal [14]. In a previous study, we demonstrated that β-endorphin is a positive regulator of glucose utilization in the insulin-deficient state via activation of the opioid μ-receptor [9]. Thus, we further investigated the effect of opioid μ-receptor activation induced by Rh2 on glucose metabolism-related gene expression. Because long-term exposure is required for activation of mRNA levels, the repeated injection of Rh2 into rats lacking insulin was investigated. We found that plasma glucose concentrations of fasting STZ-diabetic rats were significantly (P < 0.01) decreased to 17.9 ± 1.4 mmol/L after repeated treatment with Rh2 (1.0 mg/kg) three times daily for one day when compared to vehicle-treated STZ-diabetic rats (23.7 ± 1.6 mmol/L). Similar to bolus treatment, plasma glucose-lowering activity was 24.4 ± 2.1 % in STZ-diabetic rats after repeated treatments of Rh2 at the same effective dose. These results show that Rh2 has therapeutic efficacy in lowering plasma glucose after repeated treatment.

We also examined Rh2-induced change of GLUT 4 gene expression in the same group of STZ-diabetic rats. In addition, expression of b-actin mRNA or actin protein was used as an internal standard in Northern or Western blot analysis. Repeated treatment of STZ-diabetic rats with Rh2 (1.0 mg/kg) resulted in an elevated expression of mRNA for GLUT 4/β-actin in soleus muscle to a level about 1.9-fold of that observed for the vehicle-treated group; this action was abolished by pretreatment with naloxonazine (1.5 mg/kg) (Fig. [4]). Similar to the effect on mRNA, repeated treatment with Rh2 (1.0 mg/kg) elevated the protein level ratio of GLUT 4/actin in soleus muscle of STZ-diabetic rats to a level about 2.1-fold of that observed for vehicle-treated rats (Fig. [5]). An inhibitory effect of naloxonazine (1.5 mg/kg) on Rh2 (1.0 mg/kg)-induced change of GLUT 4/actin protein level was also observed (Fig. [5]). Although the direct effect of Rh2 on peripheral glucose utilization needs more study, it is reasonable to suggest that an increase in gene expression of GLUT 4 is related to activation of opioid μ-receptors, which might contribute to the observed increased glucose utilization and decreased plasma glucose in STZ-diabetic rats after repeated Rh2 treatment.

In conclusion, our results suggest that the plasma glucose-lowering action of the ginsenoside Rh2 is mediated by release of β-endorphin which, in turn, activates opioid μ-receptors, resulting in enhancement of GLUT 4 gene expression in the skeletal muscle of rats with dysfunctional pancreatic β-cells. Therefore, ginsenoside Rh2 or ginseng can probably be used as a therapeutic intervention or an attractive adjuvant for diabetic patients.

Acknowledgements

We acknowledge Dr. Chen Wan-Chung for kindly supplying us with ginsenoside Rh2. The present study was supported in part by a grant from the National Science Council (NSC 93 - 2320-B-006 - 010).

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.

Fax: +886-6-238-6548

Email: jtcheng@mail.ncku.edu.tw

References

• 1 Lopez-Candales A. Metabolic syndrome X: a comprehensive review of the pathophysiology and recommended therapy.  J Med. 2001;  32 283-300
  PubMedSearch in Google Scholar
• 2 Lee F C. Facts about ginseng, the elixir of life. Elizabeth, NJ Hollyn International Corp 1992
  Search in Google Scholar
• 3  V an Kampen J,  R obertson H,  H agg T,  D robitch R. Neuroprotective actions of the ginseng extract G115 in two rodent models of Parkinson’s disease.  Exp Neurol. 2003;  184 521-9
  CrossrefPubMedSearch in Google Scholar
• 4 Kimura M, Waki I, Tanaka O, Nagai Y, Shibata S. Pharmacological sequential trials for the fractionation of components with hypoglycemic activity in alloxan diabetic mice from ginseng radix.  J Pharmacobiodyn. 1981;  4 402-9
  PubMedSearch in Google Scholar
• 5 Yokozawa T, Kobayashi T, Oura H, Kawashima Y. Studies on the mechanism of the hypoglycemic activity of ginsenoside-Rb₂ in streptozotocin-diabetic rats.  Chem Pharm Bull. 1985;  33 869-72
  PubMedSearch in Google Scholar
• 6 Liu T P, Liu I M, Cheng J T. Improvement of insulin resistance by Panax ginseng in fructose-rich chow-fed rats.  Horm Metab Res. 2005;  37 146-51
  Thieme ConnectPubMedSearch in Google Scholar
• 7  D G, Kitts D D. Ginsenosides can inhibit proliferation and induce apoptosis in cultured leukemia and intestinal cells but effects vary according to the structure of the compounds.  Faseb J. 2003;  17 A762
  PubMedSearch in Google Scholar
• 8 Curry D L, Li C H. Stimulation of insulin secretion by beta-endorphin (1 - 27 and 1 - 31).  Life Sci. 1987;  40 2053-8
  CrossrefPubMedSearch in Google Scholar
• 9 Cheng J T, Liu I M, Tzeng T F, Tsai C C, Lai T Y. Plasma glucose-lowering effect of beta-endorphin in streptozotocin-induced diabetic rats.  Horm Metab Res. 2002;  34 570-6
  Thieme ConnectPubMedSearch in Google Scholar
• 10 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
• 11 Liu I M, Chi T C, Shiao G C, Lin M T, Cheng J T. Loss of plasma glucose lowering response to cold stress in opioid mu-receptor knock-out diabetic mice.  Neurosci Lett. 2001;  307 81-4
  CrossrefPubMedSearch in Google Scholar
• 12 Cefalu W T. Evolving strategies for insulin delivery and therapy.  Drugs. 2004;  64 1149-61
  PubMedSearch in Google Scholar
• 13 Pasternak G W. Pharmacological mechanisms of opioid analgesics.  Clin Neuropharmacol. 1993;  16 1-18
  PubMedSearch in Google Scholar
• 14 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

Professor Juei-Tang Cheng

Department of Pharmacology

College of Medicine

National Cheng Kung University

Tainan City

Taiwan 70101

R.O.C.

Fax: +886-6-238-6548

Email: jtcheng@mail.ncku.edu.tw