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Electroacupuncture attenuates hepatic lipid accumulation via AMP-activated protein kinase (AMPK) activation in obese rats
  1. Meirong Gong1,
  2. Chen Cao1,
  3. Fengli Chen1,
  4. Qian Li1,
  5. Xiaolin Bi2,
  6. Yinong Sun1,
  7. Zhen Zhan3
  1. 1Department of Acupuncture and Moxibustion, Nanjing University of Chinese Medicine, Nanjing, China
  2. 2Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
  3. 3Department of Basic Medical Sciences, Nanjing University of Chinese Medicine, Nanjing, China
  1. Correspondence to Professor Zhen Zhan, Department of Basic Medical Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, China; njzhenzhan{at}sina.com

Abstract

Background Electroacupuncture (EA) may offer an effective alternative approach for the treatment of obesity. EA mobilizes energy stores, but its effect on hepatic lipid metabolism is unknown, and the underlying mechanisms remain unclear.

Objective To examine the effect of EA on hepatic lipid accumulation in diet-induced obese (DIO) rats, and to explore potential underlying mechanisms.

Methods Male Sprague-Dawley rats were fed a normal diet (control group, n=10) or a high-fat diet (HFD) for 12 weeks to induce obesity. Those exhibiting diet-induced obesity were subdivided into two groups, one receiving EA (DIO+EA group, n=10) and one left untreated (DIO group, n=10) and observed for a further 4 weeks. Body, liver and fat pad weight were measured, and liver injury was assessed histologically as well as by measuring serum values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Hepatic triglyceride (TG) and total cholesterol were quantified by enzymatic colorimetric methods. Expression of liver AMP-activated protein kinase (AMPK), acetyl-coenzyme A carboxylase (ACC), and carnitine palmitoyltransferase (CPT-1) was measured by Western blotting.

Results EA treatment led to a reduction in body, liver and fat pad weight in DIO rats. This was accompanied by decreases in hepatic TG and total cholesterol values, fatty droplet accumulation, and serum concentrations of ALT and AST. Furthermore, EA treatment restored phosphorylation levels of AMPK (Thr172) and ACC (Ser79) inhibited by HFD, and increased CPT-1 expression.

Conclusions EA reduces HFD-induced hepatic lipid accumulation, an effect that appears to be mediated through AMPK signalling pathways. Our results shed new light on the mechanisms by which EA may reduce obesity.

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Introduction

Obesity can lead to a wide range of complications, including diabetes, hyperlipidaemia, hypertension, and coronary heart disease, and is a significant public health problem worldwide.1 It is known that obesity is caused by the accumulation of surplus energy in the form of triglyceride (TG), which occurs when energy intake exceeds energy expenditure. The liver is an important mediator of glucose and lipid homeostasis. It plays a key role in the regulation of energy status,2 and excessive lipid accumulation in the liver is often associated with the development of obesity. AMP-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase that plays a key role in regulating energy metabolism.3 Recent studies have demonstrated the roles of AMPK activation in liver metabolism.3 ,4 When cellular energy stores decrease, AMPK is activated by phosphorylation of Thr-172. Activated AMPK in turn phosphorylates and inactivates metabolic enzymes such as acetyl-CoA carboxylase (ACC), which leads to a decrease in fatty acid synthesis and an increase in mitochondrial fatty acid oxidation via allosteric regulation of carnitine palmitoyltransferase-1 (CPT-1).5 ,6 Based on its important roles in lipid and liver metabolism, AMPK is an attractive molecular target for the treatment of obesity and fatty liver.7

Acupuncture has been widely used in China for thousands of years, and has recently become a popular complementary medical therapy in Western countries.8 Evidence suggests that electroacupuncture (EA) may be helpful in preventing and treating obesity.9 Our previous studies have shown that EA can reduce body weight, Lee's index (a marker similar to body mass index), and the weight of the fat pad in obese rats, as well as improve leptin resistance.10 It was recently reported that acupuncture is a potent protector of chronic liver damage induced by carbon tetrachloride (CCl4) in rats.11 In addition, acupuncture has been shown to display an anti-fibrotic role in a rat model of CCl4-induced hepatic fibrosis.12 Moreover, Tominaga et al13 found that EA increased the activity of AMPK in skeletal muscle, leading to increased fatty acid oxidation and reduced glyceride synthesis. Therefore, AMPK may play an important role in the regulation of lipogenesis in metabolic tissues.

Based on these observations, we hypothesised that EA might inhibit hepatic lipid accumulation via activation of AMPK signalling. To address this, we characterised the effects of EA on body fat accumulation and fatty liver disease in diet-induced obese (DIO) rats, and assessed the levels of phosphorylated AMPK and ACC, and the expression of CPT-1.

Methods

Animals

Four-week-old male Sprague-Dawley rats (50–70 g) were obtained from the Medical Experimental Animals Center of Southeast University (Nanjing, China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine (reference no. SYXK (SU) 2014-0001) and all procedures were conducted in accordance with the guidelines of the National Institutes of Health Animal Care and Use Committee. Animals were housed in a room at controlled temperature (22±1°C) and humidity (50±5%) with a 12 h light/12 h dark schedule, and were kept in group cages with ad libitum access to food and water. After 7 days’ acclimation to the laboratory environment, the rats were randomly divided into two groups and fed a normal diet (ND, control group, n=10) or high-fat diet (HFD: 30% fat, 40% carbohydrate, 15.5% protein, n=40) obtained from Research Diets Inc (Shuangshi, Suzhou, China). After 12 weeks, rats in the HFD-fed group with higher body weights were assigned as DIO rats (n=20, 50%) and were randomised on the basis of body weight (using a random numbers table) into two subgroups (n=10 each). All animals were kept for a further 4 weeks, during which one group of rats received EA (DIO+EA group, n=10) and one remained untreated (DIO group, n=10).

Body weights were recorded weekly throughout the experiments. In addition, naso-anal body lengths were measured and used to calculate the Lee's index (weight0.33×1000/body length). The researchers carrying out the investigations were blinded to treatment allocation.

EA treatment

During EA treatment the rats were conscious and restrained in a plastic holder. Stainless steel needles (0.22 mm in diameter and 10 mm in length, Huatuo Brand, Suzhou Medical, Suzhou, China) were inserted bilaterally at ST36 (Zusanli), which is located at the anterior tibial muscle about 10 mm below the knee joint, and ST44 (Neiting), which is located between the second and third phalanges on the foot, lateral and distal to the second metatarsodigital joint.14 Needles were fixed to the acupuncture points using rubber cement. A Han's Acupoint Nerve Stimulator (HANS, China) was used to provide electrical impulses at an intensity of 2 mA, which produced slight twitching of the limbs. The frequency of the square waves was 2/15 Hz (2 Hz alternating with 15 Hz, each lasting for 3 s). EA treatment lasted for 15 min, and was administered in the morning three times per week for 4 weeks. Rats in the control and untreated DIO groups were restrained for 15 min without EA stimulation to standardise the experimental conditions.

Laboratory analyses

At the end of the experimental period of 16 weeks the animals were euthanased with ether. Blood was collected by cardiac puncture and maintained at room temperature for 1 h before being centrifuged at 950 g for 15 min. Serum was prepared and stored at −70°C pending analysis. The abdominal cavity was opened and the perirenal, epididymal and brown fat pads were collected and weighed. The liver was also removed and weighed, and the liver index (liver to body weight ratio) was calculated.15

Serum biochemistry

Serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by enzymatic colorimetric methods using commercial kits (C009-3 for ALT, C010-3 for AST; Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer's instructions.

Determination of cholesterol and TG levels

Lipids were extracted from 100 mg of liver tissue using chloroform and methanol and dissolved in 1% triton X-100/chloroform (v/v).16 Hepatic cholesterol and TG concentrations were measured using commercial enzymatic kits (F002-1 for cholesterol, F001-1 for TG; Jiancheng Institute of Biotechnology) according to the manufacturer's instructions.

Hepatic histology

Liver tissue was fixed in 40 g/L formaldehyde, sectioned, dewaxed in xylene, rehydrated in an alcohol gradient, stained with hematoxylin and eosin (H&E) and mounted with Permount. Oil Red O was used to stain accumulated intracellular TG. Briefly, liver tissue was embedded in tissue-freezing medium (Leica, Wetzlar, Germany); 30 µm tissue sections were prepared and rinsed with distilled deionised water and 60% isopropanol, stained with Oil Red O for 1 h, then subjected to standard H&E staining and mounted on glass slides.17 Tissue destruction and fatty changes of the liver were examined by light microscopy (Olympus Medical Systems Corp, Tokyo, Japan).

Western blotting

Frozen liver tissues were homogenised at 4°C in extraction buffer (1% Triton X-100, 100 mM Tris pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylenediaminetetraacetic acid, 10 mM sodium vanadate, 2 mM phenylmethyl sulfonylfluoride, 0.1 mg/mL aprotinin) and subjected to ultrasonication at 4°C for 30 s (Ningbo, China). Tissue homogenates were centrifuged at 12 000 g at 4°C for 30 min, supernatants collected, and protein concentrations determined using Pierce BCA (bicinchoninic acid) protein assay kits. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk/0.1% Tween-20 in Tris-buffered saline (TBS) and incubated overnight with either anti-CPT-1 (Santa Cruz Biotechnology, California, USA; 1:100 dilution), anti-phospho-AMPK (Thr172), anti-phospho-ACC (Ser79), anti-AMPK, anti-ACC, or anti-β-actin (all from Cell signalling Technology, Beverly, Massachusetts, USA; 1:1000 dilution). After washing with TBS, the membrane was incubated with horseradish peroxidase-labelled anti-rabbit immunoglobulin (Cell Signalling) for 1 h at room temperature. Blots were visualised with enhanced chemiluminescence reagent and a ChemiDoc XRS imaging system (Bio-Rad, Hercules, California, USA).

Statistical analysis

Results are presented as the mean±SD unless otherwise stated. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) V.16.0 (SPSS Inc, Chicago, Illinois, USA). Groups were compared by one-way analysis of variance followed by post-hoc Tukey's test. Values of p<0.05 were considered to be statistically significant.

Results

There was one mortality (in the DIO group). In addition, two animals (one in the DIO group and one in the DIO+EA group) were excluded from the analysis after being injured during fights.

Anthropometry

As shown in table 1, DIO rats treated for 4 weeks with EA exhibited a significantly reduced body weight compared to untreated DIO animals, and a significantly decreased Lee's index. Weights of the fat pad and liver were also significantly reduced in EA-treated animals (table 2); however, there were no significant differences in the liver index between groups. No significant differences were found between the EA-treated DIO animals and the control group for any parameters at 16 weeks.

Table 1

The effects of EA on body weight, body length and Lee's index in diet-induced obese rats

Table 2

The effects of EA on liver weight and fat deposits in diet-induced obese rats

Hepatic lipid accumulation

Feeding an HFD to rats to induce obesity led to a significant increase in concentrations of hepatic TG and total cholesterol (figure 1A, B), as well evidence of fatty infiltration in the liver (figure 1C). However, each of these effects was significantly attenuated by EA treatment.

Figure 1

(A) Total liver cholesterol (TChol), (B) hepatic triglyceride (TG), and (C) representative photomicrographs of liver tissue stained with Oil Red O (magnification ×400) in 10 healthy rats (control group, n=10) and 17 rats with diet induced obesity (DIO) left untreated (DIO group, n=8) or treated with electroacupuncture (DIO+EA group, n=9) for 4 weeks. Data are mean±SD. *p<0.05 and **p<0.01 compared with control group. #p<0.05 and ##p<0.01 compared with DIO group.

A significant elevation in serum markers of liver injury (ALT and AST) was also noted in DIO rats, as compared with the control group (figure 2A, B). This was accompanied by evidence of hepatic fat deposition and associated injury (figure 2C). Again, treatment with EA mitigated these effects, suggesting that EA can suppress HFD-induced lipotoxic liver injury.

Figure 2

Serum values of (A) alanine aminotransferase (ALT) and (B) aspartate aminotransferase (AST), and (C) representative images of haematoxylin and eosin stained liver tissue (magnification ×400) (C) in 10 healthy rats (control group, n=10) and 17 rats with diet induced obesity (DIO) left untreated (DIO group, n=8) or treated with electroacupuncture (DIO+EA group, n=9) for 4 weeks. Data are mean±SD. **p<0.01 compared with control group. ##p<0.01, compared with DIO group.

AMPK signalling in the liver

Figure 3 shows the expression levels of p-AMPK, p-ACC and CPT-1 in hepatic tissues. Phosphorylation of AMPK and ACC as well as the expression of CPT-1 was suppressed in DIO animals, compared with control animals fed with ND. However, these effects were reversed following EA treatment. Compared to untreated DIO rats, EA increased expression of p-AMPK and p-ACC by approximately 1.5-fold and 3.8-fold, respectively, and CPT-1 by about 1.7-fold.

Figure 3

Representative gels demonstrating protein bands (left panel) and mean optical density expressed relative to β-actin (right panel) of key components of the AMP-activated protein kinase (AMPK) signalling pathway, measured by Western blot analysis in the liver tissues of 10 healthy rats (control group, n=10) and 17 rats with diet induced obesity (DIO) left untreated (DIO group, n=8) or treated with electroacupuncture (DIO+EA group, n=9) for 4 weeks. Data are mean±SD. **p<0.01 compared with control group. #p<0.05 and ##p<0.01 compared with DIO group. ACC, acetyl-coenzyme A carboxylase; CPT-1, carnitine palmitoyltransferase; p, phosphorylated; Ser, serine; Thr, threonine.

Discussion

Obesity is a disease resulting from excessive fat storage in the body and is one of the leading public health risk factors in both industrialised and developing countries. Drugs currently used to treat obesity, such as sibutramine, orlistat and rimonabant, can have undesirable side effects, hence there is a need to explore alternative treatments. Acupuncture is one of the oldest complementary therapies in the world.18 Acupuncture, particularly EA, has been shown to be beneficial in the treatment of obesity by reducing body weight, normalising insulin values, and improving glucose and lipid metabolism.19 However, the underlying molecular mechanisms by which EA influences glucose and lipid metabolism remain unclear. The present study was designed to explore the effects of EA on hepatic lipid accumulation in obese rats.

We observed that DIO rats had greater body mass, weight of the fat pad, and liver weight. Serum markers of liver injury (ALT and AST) were also significantly elevated, and lipid deposition and morphological changes were noted in the liver. The administration of EA not only markedly attenuated these features of obesity and fatty liver, but also reversed the elevation of TG and total cholesterol in serum. These results indicate that EA reduces HFD-induced body weight gain and liver injury by inhibiting the accumulation of fat.

AMPK has been described as an integrator of regulatory signals monitoring systemic and cellular energy status.4 Once AMPK is activated, lipogenesis in the liver is inhibited, which consequently reduces fat accumulation.20 ,21 In rats, AMPK phosphorylation has been shown to be inhibited by an HFD.22 AMPK thus appears to be a key player for cellular energy balance, and is an important target in the development of new treatments for obesity.23 Drugs used for the treatment of obesity, such as metformin and berberine, activate AMPK.24 ,25 There is also some evidence that the anti-obesity effects of EA occur via activation of AMPK.13 Here we have shown that EA restores the AMPK signalling pathway that is inhibited by an HFD, and that this is accompanied by a reduction in lipid accumulation and signs of liver damage. In the acute experiment, 10-week-old male rats (ND) received EA treatment for 7 days. Activation of AMPK was observed with p-AMPK (Thr172) values increasing by approximately 1.64-fold compared to untreated rats (unpublished data). This strongly suggests that AMPK is activated by EA, rather than by the weight loss that occurred during the 4 weeks of treatment, and that AMPK signalling mediates the hypolipidaemic effects of EA.

In summary, our data suggest that EA can induce AMPK-mediated fatty acid metabolism, leading to reduced liver damage and overall body weight. This supports that idea that EA treatment may serve as an effective complementary and alternative therapy for obesity, hyperglycaemia and fatty liver disease. Further work to understand the mechanisms of EA activation of AMPK is warranted.

Acknowledgments

The authors would like to thank Dr Changyan Ma in Nanjing Medical University for editing the manuscript.

References

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Footnotes

  • Contributors MG, ZZ and YS conceived the study and supervised its design. CC and QL contributed to the execution of the study. FC performed data management and statistical analysis. MG and XB drafted the manuscript. All authors read and approved the final manuscript.

  • Funding This work was supported by the National Natural Science Foundation of China (No. 81202742, 81403114, 81403481 and 81373747).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval This study was prospectively approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (reference no. SYXK (SU) 2014-0001) and was performed in accordance with the guidelines for animal welfare equivalent to the ‘Guide of the Use and Care of Laboratory Animals’.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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