A total of 70 Sprague-Dawley male 4-week-old rats weighing 90–100 g were provided by the Experimental Animal Center of Chongqing Medical University (medical animal certification no. SCXK-2012-0002). The approval for the present study was provided by Chongqing Medical University's Animal Care and Ethics Committee and formulated by the Ministry of Science and Technology of China. The rats were housed in a room under controlled temperature and humidity (24°C, 50%) with a 12 h light/dark cycle. The rats were provided with food and water ad libitum. After 1 week, the rats were randomly divided into normal (n=10) and IR (n=60) groups. The HFHCD composition and preparation described in a previous study was used with a few modifications (15). The HFHCD consisted of 25% fructose, 15% lard oil, 60% powdered rat chow and 50 g water per kg of diet. The IR rats were fed with HFHCD for 5 weeks. On the 6th week, the IR state was confirmed by measuring fasting plasma glucose (FPG) insulin (≥100 mg/dl), insulin sensitivity index (ISI) and IR index. All IR rats that satisfied the IR criteria were randomly divided into 4 groups, as follows: IR control group (n=15), in which the rats were fed HFHCD continuously; diet group (n=15), in which the rats were only fed a low-fat and low-carbohydrate diet (10% fructose, 5% lard oil, 85% powdered rat chow and 50 g water per kg of diet); ES group (n=15), in which the rats received ES and normal diet; and ES + diet group (n=15), in which the rats received low-fat and low carbohydrate diet and ES. All IR rats were fed for an additional 2 weeks on the experimental diets after the 5 weeks of the HFHCD and were subsequently sacrificed for the tissue collection.

The rats were positioned in a custom-made holder (patent no. ZL201420163074.2). ES was performed in bilateral Zusanli (ST36, located at the anterior aspect of the hind limb, 2 mm straight below the knee joint), Sanyinjiao (Spleen 6, located at the medial side of the hind limb, 2 mm above the tip of the medial malleolus posterior to the medial border of the tibia) and Weiwanxiashu (EX-B3, 1 mm lateral to the no. 8 thoracic spinous process) acupoints after the IR model was established. Acupuncture needles (13 mm in length and 0.22 mm in diameter) were inserted into the acupoints muscle layers to a depth of ~5 mm. The pairs of needles on the ipsilateral ST36 and SP6 acupoints were connected with the output terminals of an ES apparatus (Model SDZ-II; Suzhou Huatuo Medical Instruments Co., Ltd.). The alternating strings of dense-sparse frequencies (20 Hz for 1.05 sec and 4 Hz for 2.85 sec, alternately) were used. The intensity was adjusted to induce a slight twitch of the hind limbs (≤2 mA), with intensity lasting for 20 min/day for 14 days.

Glucose oxidase kit (Nanjing Jiancheng Taihao Biotechnology Co., Ltd. cat. no. F006) and INS ELISA kit (Thermo Fisher Scientific, cat. no. ERINS) were used. The primary antibodies were used for western blotting and immunohistochemistry as follows: Polyclonal rabbit anti-P70S6K antibody (Signalway Antibody LLC, cat. no. 21276), polyclonal rabbit anti-GSK-3 α/β antibody (Signalway Antibody LLC, cat. no. 29039), polyclonal rabbit anti-mTOR antibody (Signalway Antibody LLC, cat. no. 21214), monoclonal rabbit anti-Akt Antibody (Signalway Antibody LLC, cat. no. 48888), monoclonal rabbit anti-PI3K antibody (Signalway Antibody LLC, cat. no. 48868) and polyclonal rabbit anti-GLUT4 antibody (Abcam, cat. no. ab654). The secondary antibody for western blotting and immunohistochemistry: Goat anti-rabbit IgG secondary antibody (OriGene Technologies, Inc. cat. no. TA130015).

The rats from the various groups were anesthetized by intraperitoneally injecting 2% pentobarbital sodium (40 mg/kg). Physiological saline (250 ml, 4°C) and 4% paraformaldehyde in 500 ml of 0.1 mmol/l phosphate buffered saline (PBS; 4°C, pH of 7.4) were perfused into the left ventricle. The liver tissues, pancreas and the quadriceps femoris muscle tissues in left leg were harvested and assigned into two segments. First segments were fixed with 4% paraformaldehyde for 24–48 h (4°C) and dehydrated with 30% sucrose overnight (4°C). Second segments were used for ultrastructure observation.

To evaluate the pathologic changes and effectiveness of ES, H&E staining was performed. Liver and pancreas tissues were fixed in formalin for >24 h and then moved to 70% ethanol. Then, the tissues were dehydrated in graded alcohols, immersed in xylol and embedded in paraffin wax. The tissues were sliced into serial sections with a thickness of 6 µm each using a paraffin slicing machine and stained with H&E (0.5–1% eosin; 3.3% hematoxylin) under room temperature for 80 min. Digital images were captured using an Olympus light microscope with a ×10 objective and an Olympus camera (Olympus-45; Olympus Corporation) with a total magnification of ×100. The images were further analyzed by a blinded investigator.

For the immunohistochemical evaluation, 10 µm thick sections of the liver, pancreas and muscle tissues from each group were prepared. Briefly, endogenous peroxidase was blocked by 3% H₂O₂ for 15 min at room temperature. The sections were further washed with 0.01 mmol/l PBS and blocked with 10% horse serum (Thermo Fisher Scientific, Inc., cat. no. 31874) for 20 min at room temperature. Next, the sections were incubated with primary antibodies (PI3K, Akt, GLUT 4, GSK-3α, GSK-3β, mTOR, P70S6K; all 1:200) at 4°C overnight. The sections were rinsed with 0.01 mmol/l PBS and reacted with secondary antibodies (1:400) for 20 min at 37°C. The slides were rewashed with 0.01 mmol/l PBS. DAB horseradish peroxidase color development kit (Beyotime Institute of Biotechnology, cat. no. P0202) was used for developing at room temperature for 5–10 min. The slides were counterstained by 3.3% hematoxylin at room temperature for 2 min after the slides were rewashed with 0.01 mmol/l PBS. Lastly, the slides were mounted in 50% glycerol dissolved in 0.01 mmol/l PBS.

A portion of each specimen from the sections perfused with 4% paraformaldehyde was immediately fixed in 2.5% glutaraldehyde for 2 days at room temperature after washing with PBS. The glutaraldehyde-fixed specimens were treated in 2% osmic acid for 1 h, dehydrated in serial alcohol (50% ethanol, 75% ethanol, 95% ethanol and absolute alcohol), infiltrated with propylene oxide and embedded in araldite for morphometric analyses on semithin sections (70–90 nm). The sections were mounted on Cu grids and stained with 3% uranyl acetate for 30 min and then 2% lead citrate for 15 min at room temperature. Images were obtained by transmission electron microscopy (Hitachi-7500; Hitachi, Ltd.).

Blood was collected from the left cardiac cavity and centrifuged at 3,000 × g for 10 min at 4°C to isolate the supernatant/plasma. FPG levels were determined using a glucose oxidase kit according to the manufacturer's protocol. The plasma levels of the fasting insulin were measured by INS ELISA kit (Thermo Fisher Scientific, Inc., cat. no. ERINS). Non-esterified free fatty acids assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. A042-2-1), total cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. F002-1-1) and triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. A110-1-1) were used to measure FFA, TC and TG, respectively. The ISI was defined as follows: ISI=l g (1/FPG × serum insulin). The IR index was defined as follows: IR index=FPG level (mg/dl) × fasting serum insulin level (ng/ml)/22.5 (16).

Protein extraction was performed according to standard procedures. Briefly, samples were homogenized in 0.3 ml of disrupting RIPA lysis buffer (Beyotime Institute of Biotechnology, cat. no. P0013B) and 1% protease inhibitor (Beyotime Institute of Biotechnology, cat. no. ST506). The homogenate was centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was diluted with 5X protein loading buffer (Beyotime Institute of Biotechnology) and then heated at 95°C for 5 min. Samples containing 40 µg protein were separated by 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. Blotted membranes were blocked with 5% skimmed milk at room temperature for 4 h. For immunoblotting, the following primary antibodies were used: Monoclonal rabbit anti-PI3 kinase antibody (1:1,000), monoclonal rabbit anti-Akt antibody (1:1,000), polyclonal rabbit anti-GLUT4 antibody (1:1,000), polyclonal rabbit anti-GSK-3 α/β antibody (1:1,000), polyclonal rabbit anti-P70S6K antibody (1:1,000) and polyclonal rabbit anti-mTOR antibody (1:1,000). All the primary antibodies were incubated at 4°C overnight. The secondary antibody used was goat anti-rabbit IgG (1:500) at room temperature for 2 h. Western bands were quantified by gel densitometry (GS-900™ calibrated densitometry system, cat. no. 1707991, Bio-Rad Laboratories, Inc.). The ratio between protein and GAPDH expression for each sample was obtained (each point was repeated in triplicate).

The statistical analyses were performed using SPSS 11.0 (SPSS, Inc.). The results were expressed as the mean ± standard error of the mean. Data were compared using the Duncan Multiple Range test following one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

After being fed with HFHCD for 5 weeks, the rats demonstrated signs of IR; however, no significant changes in overall body weight were observed in the IR rats compared with the normal rats (Table I). Following the administration of specific diets, the weight of rats in the diet, ES and ES + diet group were significantly decreased compared with those in the IR control group (P<0.05; Table I). The weight of rats in the ES + diet group significantly decreased compared with in the diet and ES groups (P<0.05; Table I). This demonstrated that ES combined with low-carbohydrate, low-fat diet can reduce weight significantly. The HFHCD caused IR, as indicated by a significant increase in FPG, insulin, IR index, TG, FFA and TC, and a decrease in ISI compared with the normal group (Table I). After 14 days, the rats in the ES + diet group demonstrated significantly lower serum levels of FPG, insulin, IR index, TG, FFA and TC compared with those in the IR control, diet and ES groups (P<0.05, n=15; Table I).

As demonstrated in Fig. 1, hepatocytes in the normal group were distributed around the central veins radially and presented cord-like arrangement. Hepatocellular nuclei were clear without inflammation and necrosis. HFHCD induced considerable hepatic damage with evident necrotic foci and inflammatory cell infiltration. The hepatic cord structure in the IR control group was destroyed and the hepatocyte arrangement became disorderly (Fig. 1). Considerable lipid droplets and cavitations were observed in hepatocytes by H&E staining. The diet treatment resulted in insignificant changes compared with the IR control group. The ES-treated group demonstrated fewer fat droplets, whereas the steatosis in the livers of the rats in the ES + diet group was improved (Fig. 1). These observations were confirmed by electron microscopy (Fig. 1), which revealed hepatocytes with abundant lipid droplets in the IR control group and few fat droplets in the diet group. In the ES and ES + diet group, inflammatory cell infiltration was improved. Lipid droplets and cavitations in the hepatocyte decreased. The hepatocyte arrangement in the ES + diet group was notably enhanced compared with that in the IR control group. These results suggested that treatment with ES and diet could ameliorate the effects of HFHCD.

The H&E-stained sections revealed that the pancreatic islet in the normal group was oval and large compared with the IR control group, thereby indicating that the pancreatic islet was injured. The pancreatic islet was improved in the ES + diet group compared with the other two treatment groups. This result suggested that ES facilitated the maintenance of the pancreatic islet cell morphology.

To investigate whether PI3K/Akt/mTOR signaling was involved with the mechanism of ES and diet therapy in insulin activity in rats, mTOR, p70S6K, PI3K and Akt expression levels were examined at the lesion sites in the different groups by immunohistochemistry and western blot analysis. As demonstrated in Fig. 2, the protein expression levels of mTOR and its downstream substrate, p70S6K, were significantly increased in the muscle tissue of IR rats fed with HFHCD (P<0.05; Fig. 2). Following treatment, the protein expression levels of mTOR and p70S6K significantly decreased in the ES + diet group compared with the other groups (P<0.05; Fig. 2).

PI3K and Akt protein expression levels were analyzed in the hepatic tissues. Western blot analysis demonstrated that the PI3K and Akt expression levels significantly decreased in the IR rats compared with the normal rats (P<0.05) (Fig. 3C and D). This result was consistent with the immunohistochemistry results (Fig. 3A and B). Following treatment, the PI3K and Akt expression levels were upregulated in the ES + diet group compared with rats in other groups (P<0.05; Fig. 3). These results suggested that the excessive nutrients hyperactivated the mTOR pathway and may have led to IR via suppression of PI3K/Akt signaling. This result was consistent with previous findings (14). ES and diet treatment may have improved IR by mTOR inhibition and PI3K/Akt signaling activation.

The protein expression levels of GSK-3α in liver tissue and GSK-3β in muscle tissue, which are serine kinases and identified as key negative regulators of glycogen synthesis (17), significantly increased in the IR control group compared with the normal groups (Fig. 4). Following treatment, the GSK-3α/β expression levels in the ES + diet group were significantly downregulated compared with the other groups (Fig. 4; P<0.05). Thus, ES may improve glycogen synthesis and IR by inhibiting GSK-3α/β expression (18). To detect the effect of ES and diet therapy on glycogen synthesis, the protein expression levels of GLUT4, which maintains glucose homeostasis in striated muscle (19), were also investigated. The results demonstrated that the GLUT4 protein expression levels increased significantly in the quadriceps femoris muscle tissues of the ES + diet group compared with those of the other groups (Fig. 5; P<05). This result suggested that combination treatment may improve glucose transportation by enhancing GLUT4 expression.