Diabetes 1
Diabetes 1
Diabetes 1
Original Article
Electroacupuncture at the Zusanli (ST-36) Acupoint Induces
a Hypoglycemic Effect by Stimulating the Cholinergic Nerve in
a Rat Model of Streptozotocine-Induced Insulin-Dependent
Diabetes Mellitus
Yu-Chen Lee,1, 2 Te-Mao Li,2 Chung-Yuh Tzeng,3 Ying-I Chen,4 Wai-Jane Ho,4
Jaung-Geng Lin,2 and Shih-Liang Chang2, 4
1 Department of Acupuncture, China Medical University Hospital, Taiwan
2 Schoolof Chinese Medicine, China Medical University, Taichung City, Taiwan
3 Department of Orthopedics, Taichung Veterans General Hospital, Taichung City, Taiwan
4 Department of Medicinal Botanicals and Health Care, Da-Yeh University, Chunghwa County, Taiwan
Animal studies have shown that electroacupuncture (EA) at Zusanli (ST-36) and Zhongwan (CV-12) acupoints reduces plasma
glucose concentrations in rats with type II diabetes. However, whether EA reduces plasma glucose levels in type I diabetes is still
unknown. In this study, we explore the various non-insulin-dependent pathways involved in EA-induced lowering of plasma
glucose. Streptozotocin (STZ) (60 mg kg−1 , i.v.) was administered via the femoral vein to induce insulin-dependent diabetes
in non-adrenalectomized and in adrenalectomomized rats. EA (15 Hz) was applied for 30 min to bilateral ST-36 acupoints
after administration of Atropine (0.1 mg kg−1 i.p.), Eserine (0.01 mg kg−1 i.p.), or Hemicholinium-3 (5 μg kg−1 i.p.) in non-
adrenalectomized rats. Rats administered acetylcholine (0.01 mg kg−1 i.v.) did not undergo EA. Adrenalectomized rats underwent
EA at bilateral ST-36 acupoints without further treatment. Blood samples were drawn from all rats before and after EA to measure
changes in plasma glucose levels. Expression of insulin signaling proteins (IRS1, AKT2) in atropine-exposed rats before and after
EA was measured by western blot. Atropine and hemicholinium-3 completely blocked the plasma glucose lowering effects of EA,
whereas eserine led to a significant hypoglycemic response. In addition, plasma glucose levels after administration of acetylcholine
were significantly lower than the fasting glucose levels. In STZ-adrenalectomized rats, EA did not induce a hypoglycemic response.
EA stimulated the expression of IRS1 and AKT2 and atropine treatment blocked the EA-induced expression of those insulin
signaling proteins. Taken together, EA at the ST-36 acupoint reduces plasma glucose concentrations by stimulating the cholinergic
nerves.
by inducing secretion of endogenous β-endorphin. Studies a hypoglycemic effect in insulin-dependent types is not well
have also shown that stimulating the CV-12, CV-4 and understood.
ST-36 acupoints with a specific frequency on both sides Although acupuncture is not suitable as a long-term
of rats significantly reduces their plasma glucose levels [5, treatment for diabetes, it may be suitable as a short-
11]. Stimulation of acupoints results in significantly greater term intervention in patients with unstable plasma glu-
plasma glucose lowering effects than stimulation of adja- cose control [6, 19, 20]. Previously, we explored various
cent non-acupuncture points [5]. Researches exploring this insulin-dependent pathways through which EA induces
mechanism discovered that the insulin secretion encouraged hypoglycemia [5]. In this study, we explore the various
by EA originates from endorphin stimulation, and that non-insulin-dependent pathways involved in EA-induced
sufficient amounts of the opioid receptor binding blocker lowering of plasma glucose.
naloxone (1 mg kg−1 , i.v.) inhibits the EA-induced plasma
glucose lowering effect [5]. Lin et al. showed that 15 Hz 2. Methods
EA at the CV-12 acupoint in adrenalectomized rats resulted
in reduced plasma glucose levels [12]. A relative study 2.1. Animal Models. Normal male Wistar Rats weighing
showed that a stimulation frequency of 2 Hz have a minor ∼250–350 g and aged 8–10 weeks were purchased from
plasma glucose lowering effect [13]. Lin et al. concluded that the BioLASCO animal center. After a 1-week adaptation
multiple sources of endogenous opioid peptide participate in period, animals were randomly divided into two groups.
the hypoglycemic effect of EA (15 Hz) stimulation at the CV- In Group I, insulin-dependent diabetes was induced by
12 acupoint [12]. Researchers compared the plasma glucose administration of streptozotocin (STZ) (60 mg kg−1 , i.v.) via
lowering effect of low frequency (2 Hz) EA stimulation at the femoral vein on the third day of a 3-day fasting period as
the ST-36 acupoint with that at the CV-12 acupoint and described previously [11, 21]. In Group II, rats underwent
found that the ST-36 acupoint was more effective in reducing bilateral adrenalectomy. After a 3-day recovery period,
plasma glucose levels. Studies have repeatedly observed that insulin-dependent diabetes was induced by administration
effect in adrenalectomized rats [14]. It has been shown of STZ (60 mg kg−1 , i.v.) via the femoral vein as previously
that the hypoglycemic effect of low frequency (2 Hz) EA described [12, 13]. Animals were housed in plexiglass cages
stimulation at the ST-36 acupoint in adrenalectomized rats at a constant room temperature of 22 ± 2◦ C with a relative
was not attenuated by naloxone treatment [15]. This finding humidity of 65 ± 5%. Rats were fed standard rat chow
implies that the glucose lowering effects of 2 Hz EA applied and were given free access to water. Animals were treated
to ST-36 acupoints do not involve the endogenous opioid in accordance with the National Institute of Health (NIH)
peptide mechanism only. Whether or not the parasympa- Guide for the Care and Use of Laboratory Animals, and the
thetic nervous system participates in the plasma glucose study protocol was approved by the ethics committee of the
lowering effect of EA is an interesting question. Acetylcholine China Medical University, Taichung, Taiwan.
(Ach) affects and relaxes vascular smooth muscles and lowers
blood pressure. This occurs mainly through binding to the 2.2. EA. Acupoints were located according to body-length
M3 receptor, which results in an increase in intracellular measurement as described elsewhere [22]. The ST-36 acu-
Ca2+ levels, which in turn leads to eNOS activation and point was located on the anterior tibia muscle approximately
NO production. The parasympathetic nerve blocker atropine upper 1/6 to the length of lower leg below the knee. Bilateral
blocks muscarinic receptors and directly competes with ST-36 acupoints were punctured in a vertical and deep
Ach for the M3 receptor. The nerve blocker hemicholin- manner with 1.27-cm, 32-gauge acupuncture needles. After
ium (HC-3) represses the choline re-uptake channel of a 5-min needling period, EA was performed for 30 min at
cholinergic nerves to directly repress Ach production instead a frequency of 15 Hz and an amplitude of 10 mA using a
of acting upon the nerve transmission system [16]. The HANS LY257 acupoint and nerve stimulator (Healthtronics,
Chinese scholar Zhao regulated the parasympathetic nervous Singapore).
system and eNOS expression using sheep red blood cell
(SRBC) to induce an immune response and HC-3 blocker 2.3. Plasma Glucose Assay. Animals were anesthetized using
to test the role of the parasympathetic nervous system pentobarbital (40 mg kg−1 i.p). Approximately 0.3–0.5 mL of
in immune system regulation induced by EA. Zhao used blood was obtained from a femoral vein using a 1-mL syringe
the lymphocyte transformation test (LTT) and measured containing heparin. The collected blood was introduced into
interlukin-2 (IL-2) activity to evaluate whether the EA- eppendorff tubes, lightly shaken and then stored on ice.
induced immune system regulatory effects are due to the Following centrifugation at 21 880 × g for 5 min, a Glucose
release of Ach by parasympathetic nerves [17]. Hsieh et al. UV reagent (Raichem, USA) was added to test the amount of
tested the effects of low frequency (2 Hz) and high frequency biological index glucose contained in the serum. The content
(100 Hz) EA stimulation at the ST-36 acupoint on heart was measured using a fully automatic biochemical analyzer
rate and skin temperature in humans and found that high- (Roche COBAS-MIRA-PLUS, USA).
frequency EA stimulation led to a reduction in heart rate
by activating parasympathetic nerves [18]. Previous studies 2.4. Experimental Protocol
have shown that EA has plasma glucose lowering effects in
non-insulin-dependent types; however, the plasma glucose 2.4.1. Electrical Acupuncture Hypoglycemic Experiment. STZ-
lowering effects and the mechanisms by which EA induces induced diabetic rats (n = 16) were randomly divided into
Evidence-Based Complementary and Alternative Medicine 3
100
2.4.2. Examination of EA Impact on Glucose Mechanisms.
Atropine Experiment: Atropine 0.1 mg kg−1 was injected into 0
the abdomens of 12 STZ-induced diabetic rats 30 min prior Non-EA EA
to the experiment. The 12 rats were randomly and equally
divided into the EA group or the control group. Blood was Before
extracted for glucose testing prior to the experiment and After
30 min after the experiment. Figure 1: Hypoglycemic effects of ST-36 EA in STZ rats: EA
Eserine Experiment: Eserine 0.01 mg kg−1 was injected represents fasting for 12 h and EA treatment for 30 min; non-
into the abdomens of 12 STZ-induced diabetic rats 30 min EA represents the non-EA control group; comparison of plasma
prior to the experiment. The rats were then randomly glucose levels was performed by self-pair t-test, ∗∗ P < .01.
divided into the EA group (N = 6) or the control group
(N = 6). Blood was extracted for glucose testing immediately
before and 30 min after the experiment.
temperature and incubated with specific primary antibodies
Hemicholinium (HC-3) Experiment: HC-3 (5 μg kg−1 )
(Santa Cruz Biotechnology, Inc., CA, USA). After washing
was injected into the abdomens of 12 STZ-induced diabetic
the membranes in a buffer containing 0.1% Tween 20 in
rats 30 min prior to the experiment. The 12 rats were then
1 × PBS, blots were incubated with a horseradish peroxidase-
randomly and equally divided into the EA group or the
linked specific second antibody (Santa Cruz Biotechnology,
control group. Blood was extracted for glucose testing 30 min
Inc.) followed by enhanced chemiluminescence detection
before and 30 min after the experiment.
using ECL reagent plus (PerkinElmer Life Sciences, Inc.,
Ach Experiment: This experiment was performed on
USA). Band intensities were quantified by densitometry to
six STZ-induced diabetic rats. Blood was extracted from
observe the target proteins. β-actin served as a loading
anesthetized animals 30 min prior to the administration of
control.
Ach 0.01 mg kg−1 i.v. EA was not performed. Blood was
extracted for glucose testing 0, 30 and 60 min after the
beginning of the experiment. 2.5. Statistical Analysis. The experimental results for each
EA in Adrenalectomized Rats: A total of 12 STZ-induced group are expressed in means ± SEM; t-test and ANOVA
diabetic rats that had undergone adrenalectomy were ran- statistical analyses were performed. Statistically significant
domly divided into the EA group (N = 6) or the control differences were set at P < .05.
group without EA (N = 6). Blood was extracted for glucose
testing 30 min before and 30 min after the experiment. 3. Results
2.4.3. Western Blot Assay. Fasting STZ rats (n = 12) were 3.1. Hypoglycemic Effect. EA (15 Hz) was applied to bilateral
randomly divided into EA or non-EA groups. The other ST-36 acupoints in eight STZ-induced diabetic rats. As seen
fasting STZ rats (n = 12) were randomly divided into the in Figure 1, there was a significant decrease in mean plasma
EA or non-EA group 30 min after treatment with atropine glucose levels before EA (418 ± 82 mg dL−1 ) and 30 min after
0.1 mg kg−1 , i.p. At the end of treatment (30 min) in EA (372 ± 77 mg dL−1 ) (n = 8, P < .01). There were no
each group, portions of the gastrocnemius muscles were significant differences in mean plasma glucose levels in the
taken as samples for analysis of insulin signaling proteins control (non-EA) group (range 447 ± 69 to 420 ± 61 mg dL−1 ;
(IRS1, AKT2). The samples were homogenized in buffer P > .05).
solution before centrifugation at 16 440 × g. The obtained
supernatant was used to estimate the amount of protein 3.2. The Influence of Atropine, HC-3 and Eserine. Atropine
using an assay kit (Bio-Rad Laboratories, CA, USA). The (0.1 mg kg−1 i.p.) and HC-3 (5 μg kg−1 i.p.) blocked the
supernatant (protein) was added to a 4× loading dye and hypoglycemic effects of 15 Hz EA performed on the ST-
boiled for 15 min at 95◦ C for denaturing. This process 36 acupoint. Thirty minutes following treatment, mean
produced a separating (8%) and stacking gel. Then, protein plasma glucose levels were 413 ± 29 mg dL−1 in the atropine
(90 μg mL−1 ) in the buffer solution was loaded into each group and 510 ± 109 mg dL−1 in the HC-3 group. After EA
well for electrophoresis. Proteins were electrophoretically stimulation, mean plasma glucose levels had decreased by
transferred to polyvinylidene difluoride membranes at 4◦ C. 1 ± 4% to 408 ± 32 mg dL−1 in the atropine group and by 3 ±
The membranes were then blocked with 5% nonfat dry 7% to 494 ± 106 mg dL−1 in the HC-3 group. There were no
milk in phosphate buffered saline (PBS) for 1 h at room significant differences before and after EA treatment in either
4 Evidence-Based Complementary and Alternative Medicine
Table 1: Influence of atropine, eserine and HC-3 on plasma glucose Table 2: Hypoglycemic response to EA in STZ-adrenalectomized
levels in STZ rats that underwent EA treatment at ST-36. rats.
Group (n = 6) Before After HGA% Group (n = 6) Before After HGA%
Atropine + EA 413 ± 29 408 ± 32 –1 ± 4 ADX-EA 507 ± 39 494 ± 63 –3 ± 6
Atropine 480 ± 51 480 ± 54 0±2 ADX-non EA 460 ± 75 446 ± 78 –3 ± 3
Eserine + EA 406 ± 55 331 ± 57∗∗ –19 ± 6# The level of plasma glucose are expressed as mean ± SEM (mg dL−1 ), n:
Eserine 460 ± 111 417 ± 105 –9 ± 6 sample number; ADX-non-EA: adrenal glands were removed + STZ induced
but did not undergo EA; ADX-EA: adrenal glands were removed + STZ
HC-3 + EA 510 ± 109 494 ± 106 –3 ± 7 induced, followed by EA; before: before EA; after: after EA treatment at ST-
HC-3 491 ± 61 471 ± 50 –4 ± 4 36; comparison of plasma glucose levels before and after EA by self-pair t-
The levels of plasma glucose are expressed as mean ± SEM (mg dl−1 ), test statistical analysis.
n: sample number; atropine: atropine 0.1 mg kg−1 i.p. treatment;
atropine + EA: atropine 0.1 mg kg−1 i.p. treatment prior to EA; eserine + EA:
eserine 0.01 mg kg−1 i.p. treatment prior to EA; HC-3: HC-3, 5 μg kg−1 Actin
i.p. treatment; Before: plasma glucose levels 30 min following medicinal
treatment; After: plasma glucose levels following 30 min of EA treatment 1.2
to ST-36; HGA%: hypoglycemic activity; comparison of before and after ∗∗ ∗∗
EA treatment by self-pair t-test ∗∗ P < .01 versus before; Student’s t-test 1
# P < .05 versus the control group.
0.8
Signal
0.6
group. In addition, there were no significant differences in
mean plasma glucose levels after injection with atropine or 0.4
HC-3 in the control group (P > .05) (Table 1). Conversely,
15 Hz EA stimulation in STZ-induced diabetic rats treated 0.2
with eserine (0.01 mg kg−1 i.p.) led to a reduction in mean
0
plasma glucose levels. Mean glucose levels were 406 ± STZ STZ + Atro STZ STZ + Atro
55 mg dL−1 following eserine treatment and 331±57 mg dL−1 IRS-1 AKT2
30 min after EA treatment, representing a decrease of 19 ± 6%
(n = 6). The difference in mean values before and after EA Non EA
was significant (P < .01). EA
[4] M. Chao, D. Zou, Y. Zhang et al., “Improving insulin resistance [20] H.-C. Pai, C.-Y. Tzeng, Y.-C. Lee et al., “Increase in plasma
with traditional Chinese medicine in type 2 diabetic patients,” glucose lowering action of rosiglitazone by electroacupuncture
Endocrine, vol. 36, pp. 268–274, 2009. at bilateral Zusanli acupoints (ST.36) in rats,” Journal of
[5] S. L. Chang, J. G. Lin, T. C. Chi, I. M. Liu, and J. T. Acupuncture and Meridian Studies, vol. 2, no. 2, pp. 147–151,
Cheng, “An insulin-dependent hypoglycaemia induced by 2008.
electroacupuncture at the Zhongwan (CV12) acupoint in [21] J.-T. Cheng, I.-M. Liu, T.-C. Chi, T.-F. Tzeng, F.-H. Lu, and
diabetic rats,” Diabetologia, vol. 42, no. 2, pp. 250–255, 1999. C. J. Chang, “Plasma glucose-lowering effect of tramadol in
[6] M. Y. Shapira, E. Y. Appelbaum, B. Hirshberg, Y. Mizrahi, streptozotocin-induced diabetic rats,” Diabetes, vol. 50, no. 12,
H. Bar-On, and E. Ziv, “A sustained, non-insulin related, pp. 2815–2821, 2001.
hypoglycaemic effect of electroacupuncture in diabetic Psam- [22] V. V. Romita, K. Yashpal, C. W. Hui-Chan, and J. L. Henry,
momys obesus,” Diabetologia, vol. 43, no. 6, pp. 809–813, “Intense peripheral electrical stimulation evokes brief and
2000. persistent inhibition of the nociceptive tail withdrawal reflex
[7] Y. Fukazawa, T. Maeda, and S. Kishioka, “The pharmacolog- in the rat,” Brain Research, vol. 761, pp. 192–202, 1997.
ical mechanisms of electroacupuncture,” Current Opinion in [23] H. J. Lee, The impact of acupuncture on diabetic mice, M.S.
Investigational Drugs, vol. 10, no. 1, pp. 62–69, 2009. thesis, China Medical University Graduate Institute of Chinese
[8] C. V. Rizos, M. S. Elisaf, D. P. Mikhailidis, and E. N. Medicine, Taichung, China, 1982.
Liberopoulos, “How safe is the use of thiazolidinediones in [24] J. M. Santos, S. B. Ribeiro, A. R. Gaya, H.-J. Appell, and J.
clinical practice?” Expert Opinion on Drug Safety, vol. 8, no. A. Duarte, “Skeletal muscle pathways of contraction-enhanced
1, pp. 15–32, 2009. glucose uptake,” International Journal of Sports Medicine, vol.
[9] O.-P. R. Hamnvik and G. T. McMahon, “Balancing risk and 29, no. 10, pp. 785–794, 2008.
benefit with oral hypoglycemic drugs,” Mount Sinai Journal of [25] D. G. Patel, “Role of parasympathetic nervous system in
Medicine, vol. 76, no. 3, pp. 234–243, 2009. glucagon response to insulin-induced hypoglycemia in normal
[10] K. W. Chen, T. Liu, H. Zhang, and Z. Lin, “An analytical and diabetic rats,” Metabolism, vol. 33, no. 12, pp. 1123–1127,
review of the Chinese literature on Qigong therapy for diabetes 1984.
mellitus,” American Journal of Chinese Medicine, vol. 37, no. 3, [26] S. R. Barry and A. Gelperin, “Acetylcholine turnover in
pp. 439–457, 2009. an autoactive molluscan neuron,” Cellular and Molecular
[11] S.-L. Chang, K.-J. Lin, R.-T. Lin, P.-H. Hung, J.-G. Lin, and J.- Neurobiology, vol. 4, no. 1, pp. 15–29, 1984.
T. Cheng, “Enhanced insulin sensitivity using electroacupunc- [27] K. Y. Liu, Y.-C. Wu, I.-M. Liu, W. C. Yu, and J.-T. Cheng,
ture on bilateral Zusanli acupoints (ST 36) in rats,” Life “Release of acetylcholine by syringin, an active principle of
Sciences, vol. 79, no. 10, pp. 967–971, 2006. Eleutherococcus senticosus, to raise insulin secretion in Wistar
[12] J.-G. Lin, W.-C. Chen, C.-L. Hsieh et al., “Multiple sources rats,” Neuroscience Letters, vol. 434, no. 2, pp. 195–199, 2008.
of endogenous opioid peptide involved in the hypoglycemic [28] K. Nonogaki, “New insights into sympathetic regulation of
response to 15 Hz electroacupuncture at the Zhongwan acu- glucose and fat metabolism,” Diabetologia, vol. 43, no. 5, pp.
point in rats,” Neuroscience Letters, vol. 366, no. 1, pp. 39–42, 533–549, 2000.
2004. [29] S. S. Castillo, “A possible role of insulin-like growth factor-II
[13] J. G. Lin, S. L. Chang, and J. T. Cheng, “Release of beta- C-peptide in regulating the function of steroidogenic cells in
endorphin from adrenal gland to lower plasma glucose adult frog adrenal glands,” Acta Histochemica, vol. 110, no. 6,
by the electroacupuncture at Zhongwan acupoint in rats,” pp. 451–461, 2008.
Neuroscience Letters, vol. 326, pp. 17–20, 2002. [30] T. T. Yang, I. M. Liu, H. T. Wu, and J. T. Cheng, “Mediation
[14] S. L. Chang, J. G. Lin, C. L. Hsieh, and J. T. Cheng, of protein kinase C zeta in mu-opioid receptor activation
“Comparision of hypoglycemic effect in different acupoints for increase of glucose uptake into cultured myoblast C2C12
response to 2 Hz electroacupuncture,” Journal of Chinese cells,” Neuroscience Letters, vol. 465, pp. 177–180, 2009.
Medicine, vol. 13, pp. 111–117, 2002.
[15] S.-L. Chang, C.-C. Tsai, J.-G. Lin, C.-L. Hsieh, R.-T. Lin, and
J.-T. Cheng, “Involvement of serotonin in the hypoglycemic
response to 2 Hz electroacupuncture of zusanli acupoint
(ST36) in rats,” Neuroscience Letters, vol. 379, no. 1, pp. 69–
73, 2005.
[16] W.-K. Lee, S.-T. Kao, I.-M. Liu, and J.-T. Cheng, “Increase of
insulin secretion by ginsenoside Rh2 to lower plasma glucose
in Wistar rats,” Clinical and Experimental Pharmacology and
Physiology, vol. 33, no. 1-2, pp. 27–32, 2006.
[17] X. Zhao, “Effect of HC-3 on electroacupuncture-induced
immunoregulation,” Zhen Ci Yan Jiu, vol. 20, pp. 59–62, 1995.
[18] C.-L. Hsieh, J.-G. Lin, T.-C. Li, and Q.-Y. Chang, “Changes of
pulse rate and skin temperature evoked by electroacupuncture
stimulation with different frequency on both zusanli acu-
points in humans,” American Journal of Chinese Medicine, vol.
27, no. 1, pp. 11–18, 1999.
[19] R.-T. Lin, C.-Y. Tzeng, Y.-C. Lee et al., “Acute effect of
electroacupuncture at the Zusanli acupoints on decreasing
insulin resistance as shown by lowering plasma free fatty acid
levels in steroid-background male rats,” BMC Complementary
and Alternative Medicine, vol. 9, Article ID 26, 2009.