Journal of Acupuncture and Meridian Studies
Volume 2, Issue 2 , Pages 147-151, June 2009

Increase in Plasma Glucose Lowering Action of Rosiglitazone by Electroacupuncture at Bilateral Zusanli Acupoints (ST.36) in Rats

  • Hui-Ching Pai

      Affiliations

    • Graduate Institute of Integrated Medicine, China Medical University, Taichung City, Taiwan
    • ,
  • Chung-Yuh Tzeng

      Affiliations

    • Department of Orthopedics, Taichung Veterans General Hospital, Taichung City, Taiwan
    • ,
  • Yu-Chen Lee

      Affiliations

    • Department of Acupuncture, China Medical University Hospital, Taichung City, Taiwan
    • ,
  • Chin-Hsien Chang

      Affiliations

    • Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital at Keelung, Chang Gung University College of Medicine, Taiwan
    • ,
  • Jaung-Geng Lin

      Affiliations

    • Graduate Institute of Chinese Medical Science, China Medical University, Taichung City, Taiwan
    • ,
  • Juei-Tang Cheng

      Affiliations

    • Graduate Institute of Integrated Medicine, China Medical University, Taichung City, Taiwan
    • Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
    • ,
  • Shih-Liang Chang

      Affiliations

    • Department of Medicinal Botanicals and Health Care, Da-Yeh University, Chunghwa County, Taiwan
    • School of Chinese Medical Science, China Medical University, Taichung City, Taiwan
    • Corresponding Author InformationCorresponding author. 168, University Road, Dacun, Changhua, Taiwan 51591, R.O.C.

Received 15 January 2009; accepted 7 April 2009.

Article Outline

Abstract 

Objectives

Hypoglycemia induced by electroacupuncture (EA) is due to an increase of insulin secretion and/or mediation of β-endorphin. We applied EA at the Zusanli (ST.36) acupuncture point (acupoint) in combination with rosiglitazone (TZD) administration to evaluate their effect on plasma glucose and to explore possible mechanisms of action.

Methods

Thirty six normal adult Wistar rats were randomly divided into four groups: the 0.1 mg/kg TZD group (0.1TZD), 0.1 mg/kg TZD and EA group (0.1TZD + EA), EA group, and control group. In other experiments, streptozotocin was used to induce type 2 diabetes mellitus in neonatal rats; these were then randomly divided into a 0.1TZD group, 0.1TZD + EA group, and EA group and changes in plasma glucose and insulin concentrations evaluated.

Results

A marked hypoglycemic response was observed in the normal rat 0.1TZD, 0.1TZD + EA and EA groups, with the response more significant in the 0.1TZD + EA group than in the 0.1TZD group. Among the diabetic animals, the hypoglycemic responses in the 0.1TZD + EA and EA groups were greater than in the 0.1TZD group. In both the normal and diabetic rats, insulin secretion was increased by EA or 0.1TZD + EA treatment, but not by 0.1TZD.

Conclusions

The plasma glucose lowering action of rosiglitazone was increased by EA in both normal and diabetic rats, indicating that the application of EA may enhance the hypoglycemic action of this insulin sensitizer.

Key words:  electroacupuncture , insulin , plasma glucose , rosiglitazone , type 2 diabetes mellitus

 

Back to Article Outline

1. Introduction 

The natural history of type 2 diabetes mellitus (T2DM) in the prediabetic state is characterized by insulin resistance and compensatory hyperinsulinemia and/or partially impaired secretion of insulin. Resistance to insulin is the reduction of the biological response to insulin and can occur 5 to 10 years before diabetes is diagnosed. There are many methods to reduce insulin resistance, including changing diet, exercise, medicine, and Chinese medicine, in addition to acupuncture, moxibustion, and massage 1, 2, 3. But there are limitations to any single therapy, especially for insulin sensitizers. In our experience, insulin sensitizers do not have efficacious hypoglycemic effects in the short term; they must be taken over a period of time to improve insulin resistance. According to traditional Chinese medicine theory, acupuncture regulates ‘Qi and Blood’ and likely adjusts the bioavailability of substances taken internally, influencing, for example, absorption, distribution, metabolism, or excretion of substances [4]. This characteristic could help improve insulin resistance or the utility of insulin. Thus it was hypothesized in the present study that electroacupuncture (EA) could increase the hypoglycemic action of the insulin sensitizer rosiglitazone (TZD).

Previously, opioid receptors in the pancreas were investigated for their roles in the regulation of plasma glucose 5, 6. In addition, β-endorphin enhances insulin secretion [7] and endogenous opioid peptides are increased by EA to promote insulin secretion, inducing a hypoglycemic response in animals 8, 9. This improvement of insulin sensitivity was related to the downregulation of the plasma free fatty acid (FFA) concentrations and by EA [10]. Further more, EA has been reported to effectively improve abnormal lipid metabolism and reduced fat accumulation in obese rats, the latter effect possibly closely associated with its effect in regulating the balance between leptin and insulin concentrations [11].

Wistar rats have been widely used in research since the 1920s [12] and alloxan or streptozotocin, which cause damage to pancreatic cells via free-radical cytotoxicity, are used to induce T2DM in laboratory animals [13]. Streptozotocin is commonly administered to neonatal rats to create a T2DM-like animal model 14, 15 that has been useful in understanding the complex pathogenesis of T2DM. Injection of this anticancer drug into rats during the neonatal period leads to the major features typically experienced by diabetic patients, including hyperglycemia, polyphagia, polydipsia, polyuria, and abnormal glucose tolerance [16]. This T2DM-like model has shown an insulin resistance state concurrent with damage to pancreatic islet cells [17].

During intravenous glucose tolerance tests (IVGTT), plasma glucose concentrations returned to normal values early in normal rats, while EA enhanced the hypoglycemic effect of the insulin challenge test (ICT) in streptozotocin-induced diabetic rats [2]. The insulin sensitizer, rosiglitazone, improves insulin sensitivity via activation of PPAR-γ receptors, but does not enhance the secretion of insulin [18]. Nonetheless, recent reports have shown adverse effects on the liver, fluid retention, and congestive heart failure [19]. The combination of the advantages of EA's effects on the regulation of plas ma glucose and insulin with those of an insulin sensitizer comprises a new therapy for improved handling of diabetic disorders. Thus the main purpose of this study was to evaluate the combination of EA and drug therapy involving an insulin sensitizer, rosiglitazone, for enhancing hypoglycemic effects. In addition, we studied the relative mechanisms of EA's synergism with rosiglitazone by assaying plasma insulin concentrations in normal and T2DM-like animal models.

Back to Article Outline

2. Methods 

2.1. Animal model 

Male Wistar rats, aged 8-10 weeks, were obtained from the National Laboratory Animal Center in Taiwan. T2DM was induced in 2-day-old neonatal rats by intraperitoneal (i.p.) injection of 60 mg/kg of streptozotocin (Sigma Company, St. Louis, MO, USA), as described previously 8, 16. All animals were maintained under a 12:12 hour light-dark cycle (starting at 6:00 a.m.) in a temperature controlled room (25 ± 1°C) at the Animal Center of China Medical University with food (Purina Rat Chow) and water ad libitum. Animals were treated in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.

2.2. Laboratory determinations 

Plasma glucose (mmol/L) concentrations were determined using a spectrophotometric system (COBAS System, Roche Diagnostics Ltd., Rotkreuz, Switzerland) and commercially available enzymatic kits run in duplicate. The plasma insulin concentrations were measured using an ELISA kit (Linco Res earch, St. Charles, Missouri, USA) [20]. In brief, sam ples were incubated for 2 hours at room temperature in a shaker and exposed to peroxidase-conjugate and antibodies bound to a microtitration well, a conjugate produced by reaction with 3,3′,5,5′-tetramethylbenzidine, the reaction stopped by adding 1 M sulfuric acid 50 μl, the mixture shaken to produce a colorimetric endpoint, and measured spectrophotometrically. The values obtained were in pmol of peptide per liter of plasma.

2.3. Experimental protocol 

Thirty-six normal rats were randomly divided into four groups: 0.1 mg/kg TZD group (0.1TZD), 0.1 mg/ kg TZD + EA group (0.1TZD + EA), EA group, and a control group (saline). Twenty-four T2DM-like rats were also randomly divided into three groups: 0.1TZD, 0.1TZD + EA, and EA group. All animals were fasted for 12 hours and 0.1 mg/kg TZD given by oral administration to the 0.1TZD and 0.1TZD + EA groups, with the control group was treated with a similar volume of normal saline. All experiments were performed under anesthesia using pentobarbital (40 mg/kg, i.p.).

2.4. Electroacupuncture 

In the EA group, the Zusanli acupoints were located bilaterally at the anterior tibia muscles near the knees, according to a previous study. Two acupuncture needles (0.5 inch in length and 32-gauge) were inserted 2-5 mm into the muscle layer at both Zusanli acupoints, with the positive lead on the right leg and the negative lead on the left, and the EA apparatus adjusted to 15 Hz/10 mA (Han's Healthronics Likon, Taipei, Taiwan). Blood samples were taken from the femoral vein 30 minutes after TZD or saline treatment and after following 30 minutes EA.

2.5. Statistical analysis 

The hypoglycemic activity (%) was calculated as follows: (Gi-Gt) × 100/Gi; Gi was the initial glucose concentration and Gt the concentration after treatment. All values were expressed as means ± SEM in the figures and tables. The Wilcoxon signed-rank test was applied to assess differences by treatment in each group. The Kruskal-Wallis test was applied to compare the hypoglycemic effect in two or more different treatment groups, and the Mann-Whitney test was applied to compare the differences between two independent groups. For all comparisons, a p-value less than 0.05 (two-sided) was considered statistically significant.

Back to Article Outline

3. Results 

3.1. Baseline data 

Normal, neonatal Wistar rats were successfully induced to develop T2DM using streptozotocin. The resulting T2DM-like rats had mean plasma glucose concentrations of 141.7 ± 5.0 mg/dL, higher than the concentration in normal rats (122.6 ± 2.5 mg/dL, p < 0.05), and lower plasma insulin concentrations (p < 0.05) than normal rats (Table 1). Additionally, the diabetic symptoms of polyphagia, polydipsia, and polyuria in these T2DM-like rats were significantly greater than among normal Wistar rats during the experimental period.

Table 1. The baseline data between normal Wistar and T2DM rats
Wistar (n = 19)T2DM(n = 19)p value
Glucose (mg/dL)122.6 ± 2.5141.7 ± 5.0**0.0021
Insulin (IU/L)0.069 ± 0.0080.039 ± 0.004**0.0017

Values as mean SEM; n = number of animals; T2DM rats = type 2 diabetic mellitus rats; independent groups compared using non-parametric Mann-Whitney test

** p <0.01.

3.2. Effect of combined therapy in normal rats 

The fasting plasma glucose and insulin concentrations in the four groups (n = 9) of normal rats did not differ significantly. A significant hypoglycemic response was produced by the treatments in the 0.1TZD, 0.1TZD + EA, and EA groups. Additionally, greater hypoglycemic activity (%) was observed in the 0.1TZD + EA and EA groups than in the 0.1TZD group (Table 2).

Table 2. Plasma glucose concentrations effects from EA and drug combined therapy in normal Wistar rats
Group (n = 9)Before treatmentAfter treatmentHypoglycemic activity (%)
0.1TZD125 ± 2104 ± 2*− 17 ± 2,B
0.1TZD + EA124 ± 286 ± 2*− 31 ± 2,A
EA121 ± 385 ± 3*− 29 ± 2,A
Saline (control)117 ± 7109 ± 3− 7 ± 6,C

Plasma glucose concentrations as mean ± SEM (mg/dL) after 12 hours fasting; n = 9; EA = electroacupuncture; 0.1TZD = rosiglitazone 0.1 mg/kg; non-parametric Wilcoxon signed-rank test to assess differences in the means of each group

* p <0.01; Kruskal-Wallis test to compare hypoglycemic effect of different treatment groups, followed by non-parametric Mann-Whitney test to compare difference between two independent groups, A >B>C, p <0.05.

There was no significant change observed in the control group, while the plasma insulin concentrations were elevated significantly in the 0.1TZD + EA and EA groups (p < 0.05), but remained unchanged in the 0.1TZD (p = 0.73) and saline (p = 0.46) groups (Figure 1).

  • View full-size image.
  • Figure 1. 

    Effects of EA and TZD therapy on plasma insulin concentration in normal Wistar rats. EA = electroacupuncture; 0.1TZD = rosiglitazone; non-parametric Wilcoxon signed-rank test to assess differences in means within each group, *p < 0.05.

3.3. The effect of combined therapy in T2DM-like rats 

The fasting plasma glucose and insulin concentrations in the three groups of T2DM-like rats did not differ. A marked hypoglycemic response (p < 0.05) was obtained in the 0.1TZD + EA and EA groups and the hypoglycemic activity (%) was stronger in the 0.1TZD + EA and EA groups compared with the 0.1TZD group (Table 3). Insulin secretion was also enhanced in the 0.1TZD + EA and EA groups, but not in 0.1TZD group (p < 0.05) (Figure 2).

Table 3. Plasma glucose concentrations effected by EA and drug combined therapy in T2DM rats
Group (n = 8)Before treatmentAfter treatmentHypoglycemic activity (%)
0.1TZD143 ± 5142 ± 6− 1 ± 2,B
0.1TZD + EA138 ± 1393 ± 10*− 33 ± 3,A
EA137 ± 989 ± 7*− 35 ± 5,A

Plasma glucose concentrations as mean ± SEM (mg/dL) after 12 hours of fasting, n = number of animals; EA = received electroacupuncture; 0.1TZD = received rosiglitazone; non-parametric Wilcoxon signed-Rank test to assess differences in the means of each group

* p < 0.05; Kruskal Wallis test to compare hypoglycemic effect of different treatment groups, followed by non-parametric Mann-Whitney test to compare differences by treatment within each independent group, A >B, p <0.05.

  • View full-size image.
  • Figure 2. 

    Effects of EA and TZD therapy on plasma insulin concentration in T2DM rats. EA = electroacupuncture; 0.1TZD = rosiglitazone; non-parametric Wilcoxon signed-rank test to assess differences in the means of each group, *p < 0.05.

Back to Article Outline

4. Discussion 

EA treatment was combined with an insulin sensitizer (rosiglitazone) to examine the hypoglycemic effects in normal rats and rats with streptozotocin-induced T2DM. Insulin and glucose concentrations were assayed to explore the plasma glucose lowering effects of the different treatments. Among the T2DM-like rats, higher plasma glucose and lower insulin concentrations were found at baseline than for normal Wistar rats. We considered that this early stage of streptozotocin-induced T2DM in rats indicated a major partial dysfunction of insulin secretion rather than insulin resistance. The mixed types of pathogenesis observed here in the T2DM-like models correlated closely with the early stage of disease after induction [17].

Similar to previous results, EA appeared to improve glucose regulation by stimulating insulin secretion and/or improving insulin sensitivity 2, 3, 8. The 0.1 mg/kg of TZD used here was near the recommended maximal dose (8 mg) for clinical applications to a 60-kg human adult. In normal Wistar rats, 17% hypoglycemic activity was obtained 1 hour after feeding 0.1 mg/kg TZD (Table 2). In T2DM-like rats, no obvious hypoglycemic response was observed in the 0.1TZD group (Table 3), a result possibly due to the insufficiency of insulin action in these T2DM-like rats. Because no significant change in plasma glucose was obtained in the saline group of normal rats, saline treatments in the T2DM-like rats were not performed.

Usually, insulin resistance is the main mechanism of T2DM observed in the clinic, unless the patient has β-cell function failure in the pancreas; T2DM patients are always hyperinsulinemic. In this study, the growth of T2DM-like rats showed damaged pancreatic function combined with partial insulin resistance, leading to higher plasma glucose. We considered that the pancreatic islets responded to EA for lowering the plasma glucose [8]. This supports the results obtained here; secretion of insulin was promoted by the combined treatment of EA and TZD (Figure 1). In another animal model studied in 2000, in which high fat diets administered to create insulin resistance were followed by streptozotocin administration to induce T2DM, the pathology of animals appeared more similar to clinical T2DM [21]. This model merits further testing of the study's hypothesis that combined treatment with EA and TZD may increase the plasma glucose lowering action more than treatment with EA or TZD alone.

A significant hypoglycemic response was observed in the 0.1TZD + EA and EA groups, both in normal and T2DM-like rats (Table 2, Table 3), and insulin secretion increased in both groups (Figure 1, Figure 2). According to previous a report [8], the hypoglycemic action of EA was related to endogenous β-endorphin concentrations, which enhances insulin secretion. Thus a more significant hypoglycemic response in the combined EA and TZD group was observed here compared with the TZD-only group (Table 2, Table 3). While Lin et al documented the increase in plasma opioids due to EA, the effect of opioids on insulin sensitivity in addition to TZD is still unclear; further elucidating studies are needed.

In conclusion, this study demonstrated that EA treatment increased the plasma glucose lowering action of rosiglitazone in both normal and T2DM-like rats. Thus the application of EA may enhance the hypoglycemic action of this insulin sensitizer via enhanced insulin secretion in neonatal streptozotocin-induced diabetic rats.

Back to Article Outline

Acknowledgments 

We thank the National Science Council of the Republic of China for a grant (NSC-95-2320-B-039-032), and the Taichung Veterans General Hospital and DaYeh University in Taiwan for kindly providing funding (TCVGH-DYU-988310) for this research. We also thank Miss Ying-I Chen and Xin-Ping Ku for their assistance.

Back to Article Outline

References 

  1. Lin JG, Chen WC, Hsieh CL, Tsai CC, Cheng YW, Cheng JT, et al. Multiple sources of endogenous opioid peptide involved in the hypoglycemic response to 15 Hz electroacu-puncture at the Zhongwan acupoint in rats . Neurosci Lett . 2004;366:39–42
  2. Chang SL , Lin KJ , Lin RT , Hung PH , Lin JG , Cheng JT . Enhanced insulin sensitivity using electroacupuncture on bilateral Zusanli acupoints (ST 36) in rats . Life Sci . 2006;79:967–971
  3. Shapira MY , Appelbaum EY , Hirshberg B , Mizrahi Y , Bar-On H , Ziv E . A sustained, non-insulin related, hypoglycaemic effect of electroacupuncture in diabetic Psammomys obesus . Diabetologia . 2000;43:809–813
  4. Senna-Fernandes V, França D, Moreno SF, Santos-Filho S, Rogers PA, Bernardo-Filho M, et al. The effect of “Zusanli” (ST. 36) acupuncture on the bio-availability of sodium pertechnetate in Wistar rats . Acupunct Electrother Res . 2006;31:33–44
  5. Khawaja XZ , Green IC , Thorpe JR , Titheradge MA . The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain . Biochem J . 1990;267:233–240
  6. Bruni JF , Watkins WB , Yen SS . Beta-endorphin in the human pancreas . J Clin Endocrinol Metab . 1979;49:649–651
  7. Curry DL , Bennett LL , Li CH . Stimulation of insulin secretion by beta-endorphins (1-27 & 1-31) . Life Sci . 1987;40:2053–2058
  8. Chang SL , Lin JG , Chi TC , Liu IM , Cheng JT . An insulin-dependent hypoglycaemia induced by electroacupuncture at the Zhongwan (CV12) acupoint in diabetic rats . Diabetologia . 1999;42:250–255
  9. Lin JG , Chang SL , Cheng JT . Release of beta-endorphin from adrenal gland to lower plasma glucose by the electroacupuncture at Zhongwan acupoint in rats . Neurosci Lett . 2002;326:17–20
  10. Liao YY , Seto K , Saito H , Kawakami M . Effects of acupuncture on the citrate and glucose metabolism in the liver under various types of stress . Am J Chin Med . 1980;8:354–366
  11. Wang SJ , Xu HZ , Xiao HL . Effect of high-frequency electroacupuncture on lipid metabolism in obesity rats . Zhen Ci Yan Jiu . 2008;33:154–158 [In Chinese]
  12. Li CR , You XF , Jiang JD . Advance in diabetes animal models . Chinese Journal of Comparative Medicine . 2005;15:59–63 [In Chinese]
  13. Kodama T , Iwase M , Nunoi K , Maki Y , Yoshinari M , Fujishima M . A new diabetes model induced by neonatal alloxan treatment in rats . Diabetes Res Clin Pract . 1993;20:183–189
  14. Blondel O , Bailbe D , Portha B . Relation of insulin deficiency to impaired insulin action in NIDDM adult rats given streptozocin as neonates . Diabetes . 1989;38:610–617
  15. Bonner-Weir S , Trent DF , Honey RN , Weir GC . Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia . Diabetes . 1981;30:64–69
  16. Takada J, Machado MA, Peres SB, Brito LC, Borges-Silva CN, Costa C, et al. Neonatal streptozotocin-induced diabetes mellitus: a model of insulin resistance associated with loss of adipose mass . Metabolism . 2007;56:977–984
  17. Trent DF , Fletcher DJ , May JM , Bonner-Weir S , Weir GC . Abnormal islet and adipocyte function in young B-cell-deficient rats with near-normoglycemia . Diabetes . 1984;33:170–175
  18. Ciaraldi TP , Gilmore A , Olefsky JM , Goldberg M , Heidenreich KA . In vitro studies on the action of CS-045, a new antidiabetic agent . Metabolism . 1990;39:1056–1062
  19. Yki-Jarvinen H . Thiazolidinediones . N Engl J Med . 2004;351:1106–1118
  20. Porstmann T , Kiessig ST . Enzyme immunoassay techniques. An overview . J Immunol Methods . 1992;150:5–21
  21. Reed MJ, Meszaros K, Entes LJ, Claypool MD, Pinkett JG, Gadbois TM, et al. A new rat model of type 2 diabetes: the fat-fed, streptozotocin-treated rat . Metabolism . 2000;49:1390–1394

PII: S2005-2901(09)60047-9

doi:10.1016/S2005-2901(09)60047-9

Journal of Acupuncture and Meridian Studies
Volume 2, Issue 2 , Pages 147-151, June 2009

Article Tools

* Image Usage & Resolution