Type 2 diabetes mellitus (T2DM) accounts for nearly 95% of total diabetic patients worldwide [
Glucose transporter 4 (GLUT4) is highly expressed in major tissues responding to insulin including skeletal muscle, liver, and adipose tissue [
Acupuncture is proved to have certain effect on T2DM and was thereby recommended by World Health Organization (WHO) as possible therapeutic method for the disease [
According to traditional Chinese medicine (TCM), weiwanxiashu (EX-B3) is specialized in treatment of diabetes and is believed to have outstanding effect [
This study aims at observing EA weiwanxiashu’s (EX-B3’s) effects on hyperglycemia and insulin resistance of high fat diet combined with STZ-induced T2DM rats and exploring their relation with skeletal muscle GLUT4 protein expression.
Experiment animals are 65 clean level male Sprague-Dawley rats (
All the rats were fed with ordinary rodent chow for 1 week for adaptation. After that, 12 rats were selected randomly according to body weight as normal control group (fed with ordinary rodent chow). Other animals (53 rats) were prepared as T2DM model rats by high fat diet combined with STZ injection according to previous researches [
Random blood glucose (RBG) was measured 72 h after the injection, and oral glucose tolerance test (OGTT) was conducted 14 days after the injection. Animals with RBG > 16.7 mmol/L and OGTT’s 2 h time-point blood glucose > 11.1 mmol/L were selected as successfully prepared T2DM models. Altogether 36 rats were included as model animals.
During the 14 days between STZ injection and model evaluation, rats except those of the normal control group were fed with high fat diet.
According to fasting blood glucose (FBG) measured 14 days after STZ injection, T2DM model rats were divided by random block design into 3 groups. They are T2DM model group (
Rats in normal control group were nondiabetic normal rats. Rats in T2DM model group were T2DM model rats. Rats in both groups received no EA intervention. By comparing results between normal control group and T2DM model group, the study verifies the quality of T2DM model animals. By comparing results between T2DM model group and EA weiwanxiashu (EX-B3) group, the study observes EA weiwanxiashu’s (EX-B3’s) intervention effects on FBG and glucose tolerance and explores the mechanism of the effects. Sham EA group was set to clarify the genuine effects of EA, when compared with EA weiwanxiashu (EX-B3) group.
Experimental procedures.
Fixation of animals, point locating, needle insertion, and EA device connection on all rats during the 4-week intervention were done by the same persons, respectively.
FBG was tested 1 day before the intervention and at the 7th, the 14th, the 21st, and the 28th day of the intervention using enzyme end-point method [
OGTT was tested one day before the intervention and the day after the last intervention, to determine the effect of EA on rats’ glucose tolerance. Protocol: All animals were fasted overnight from 22:00 to 8:00. After the test of FBG, rats were intragastrically given glucose solution at 2 g/kg body weight [
After sacrifice of rats, same part of the quadriceps femoris of all rats was collected for western blot test of GLUT4 protein expression. Results were shown as the ratio to
For each sample, 100 mg of the tissue was ground in a 1.5 mL grinder with 1 mL radioimmunoprecipitation (RIPA) lysis buffer and protein inhibitor mixture in ice bath. The homogenate was then collected in a 1.5 mL centrifuge tube, placed on ice for 30 min (vortexed every 5 min), and then centrifuged (4°C, 10000 rpm, 15 min). The liquid supernatant was then collected for measurement of protein concentration with Bicinchoninic acid (BCA) kit, prepared at a balanced concentration of 4.67
For western blot test, polyacrylamide gel electrophoresis (80 V, 30 min for stacking gel, and 120 V, 60 min for separating gel) was made using 10% separating gel. The sample volume is 15
After sacrifice of rats, same part of the quadriceps femoris of all rats was collected. Membrane proteins were extracted with Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, LOT: OJ186747A). Other procedures are the same as protocol of western blot test listed above. Results were shown as the ratio to
Comparative ratio of skeletal muscle membrane GLUT4 expression was shown as the ratio of skeletal muscle membrane GLUT4 expression to GLUT4 expression of the whole cell.
After sacrifice of rats, the same part of the quadriceps femoris of all rats was collected for western blot test of PI3K (Y607) protein expression. Protein were extracted with phosphatase inhibitor. Other procedures are the same as protocol of western blot test listed above (PI3K polyclonal antibody purchased from Abcam, LOT: 194710-8). Results were shown as the ratio to
SPSS 19.0 was applied for the processing of data. Results were shown as
Results show that, before intervention, FBG of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group are significantly higher than that of normal control group (
EA’s intervention effect on FBG (
Groups | | FBG before intervention | FBG at the 7th day | FBG at the 14th day | FBG at the 21st day | FBG after intervention |
---|---|---|---|---|---|---|
Normal control group | 12 | 4.95 | 4.29 | 4.93 | 6.16 | 5.24 |
T2DM model group | 9 | 17.04 | 17.30 | 15.88 | 28.46 | 27.42 |
EA weiwanxiashu (EX-B3) group | 10 | 20.33 | 13.91 | 18.38 | 19.88 | 18.95 |
Sham EA group | 9 | 16.88 | 16.73 | 17.63 | 22.34 | 25.07 |
Note: △△ for
Results show that, before intervention, AUC of OGTT of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group are significantly higher than that of normal control group (
EA’s intervention effect on AUC of OGTT (
Group | | AUC of OGTT before intervention | AUC of OGTT after intervention |
---|---|---|---|
Normal control group | 12 | 14.89 ± 0.62 | 15.55 ± 0.57 |
T2DM model group | 9 | 50.38 ± 3.55 | 63.14 ± 1.09 |
EA weiwanxiashu (EX-B3) group | 10 | 52.27 ± 2.76 | 49.37 ± 5.52 |
Sham EA group | 9 | 55.50 ± 2.33 | 59.84 ± 1.76 |
Note: △△ for
Results show that, after intervention, HOMA-IR of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group is greatly higher than that of the normal control group (
EA’s intervention effect on HOMA-IR and HOMA-B (
Group | | Fasting insulin (mmol/L) | HOMA-IR | HOMA-B |
---|---|---|---|---|
Normal control group | 12 | 18.24 ± 0.65 | 4.22 ± 0.14 | 236.41 ± 28.96 |
T2DM model group | 9 | 17.48 ± 0.81 | 21.32 ± 1.62 | 15.08 ± 1.18 |
EA weiwanxiashu (EX-B3) group | 10 | 16.01 ± 0.80 | 13.51 ± 2.20 | 72.65 ± 48.07 |
Sham EA group | 9 | 15.94 ± 1.00 | 17.46 ± 0.63 | 15.39 ± 1.68 |
Note:
Results show that T2DM model animals are of remarkably lowered GLUT4 protein expression in skeletal muscle (
EA’s intervention effect on skeletal muscle GLUT4 protein expression (
Group | | GLUT4 | Membrane GLUT4 | Comparative ratio of membrane GLUT4 |
---|---|---|---|---|
Normal control group | 3 | 2.53 | 0.50 | 0.20 |
T2DM model group | 3 | 2.21 | 0.17 | 0.08 |
EA weiwanxiashu (EX-B3) group | 3 | 2.10 | 0.29 | 0.14 |
Sham EA group | 3 | 2.32 | 0.22 | 0.09 |
Note: △△ for
Membrane GLUT4 expression of the normal control group is significantly higher than T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group (
Comparative ratio of skeletal muscle membrane GLUT4 of T2DM model animals is greatly lower than normal control group (
Skeletal muscle PI3K (Y607) protein expression of T2DM model group is significantly lower than that of the normal control group (
EA’s intervention effect on skeletal muscle PI3K (Y607) protein expression (
Group | | PI3K (Y607) |
---|---|---|
Normal control group | 3 | 0.68 ± 0.02 |
T2DM model control group | 3 | 0.59 ± 0.00 |
EA weiwanxiashu (EX-B3) group | 3 | 1.25 ± 0.02 |
Sham EA group | 3 | 0.63 ± 0.03 |
Note:
According to TCM, back-
Results of our study proved that EA weiwanxiashu (EX-B3) can significantly reduce model rats’ highly rocketed FBG (Table
EA’s intervention effect on FBG. Note: △△ for
EA’s intervention effect on AUC of OGTT. Note: △△ for
EA’s intervention effect on HOMA-IR. Note: △△ for
EA’s intervention effect on HOMA-B. Note: △△ for
The above changes can be performed by activation of GLUT4. In static state, GLUT4 is sequestered in intracellular vesicles in skeletal muscle cells and has no biological activity. When stimulated by insulin signal or muscle contraction [
Previous researches show that it is possible to regulate GLUT4 expression by EA [
Our research results show that EA weiwanxiashu (EX-B3) can significantly improve hyperglycemia and insulin resistance of model rats and greatly improve skeletal muscle membrane GLUT4 expression but not cytoplasmic GLUT4 expression, indicating that EA can stimulate GLUT4 membrane translocation to reduce FBG and relieve insulin resistance. Test of skeletal muscle PI3K (Y607) protein expression demonstrates that, in our study, upregulation of GLUT4 membrane translocation is related to phosphorylation of PI3K.
This is in accordance with previous research conclusion that insulin signal takes effects in the skeletal muscle by binding insulin with the alpha-subunits of insulin receptor on the surface of cell. After that, beta-subunit of insulin receptor will autophosphorylate and lead to activating tyrosine phosphorylation of insulin receptor substrate (IRS; in skeletal muscle, it is mainly IRS-1) [
Since in our study, EA weiwanxiashu (EX-B3) group exceeds sham EA group in regulation of skeletal phosphorylated PI3K expression (Table
EA’s intervention effect on skeletal muscle GLUT4 protein expression. Note: △△ for
EA’s intervention effect on skeletal muscle membrane GLUT4 protein expression. Note: △△ for
Comparative ratio of membrane GLUT4. Note: △△ for
EA’s intervention effect on skeletal muscle PI3K (Y607) protein expression. Note: △ for
EA weiwanxiashu (EX-B3) is especially useful for intervention of T2DM.
EA weiwanxiashu (EX-B3) can upregulate skeletal muscle phosphorylated PI3K protein expression, to stimulate membrane translocation of GLUT4 and thereby increase skeletal muscle glucose intake to reduce blood glucose and relieve insulin resistance.
The authors declare that they have no competing interests.
Bing-Yan Cao and Rui Li designed the study. Huan-Huan Tian made literature review. Researchers that assisted with the experimental work are Xiao-Gang Hu (fixing the animals), Ning Jia (point locating), Yan-Jia Ma (needle insertion), and Yue-Ying Wang (EA device connection). Data collection and analysis were done by Bing-Yan Cao. All authors read and approved the final paper.
The study was funded by Ministry of Education of the People’s Republic of China (no. 313010).