Duodenal mucosal ablation with irreversible electroporation reduces liver lipids in rats with non-alcoholic fatty liver disease

Abstract

BACKGROUND

Duodenal mucosal ablation (DMA) using irreversible electroporation (IRE) with a glucagon-like peptide-1 receptor agonist has been clinically shown to reduce liver lipid deposition in non-alcoholic fatty liver disease (NAFLD). However, the specific metabolic contributions of DMA using IRE in NAFLD remain unclear.

AIM

To assess the feasibility and effectiveness of DMA using IRE in NAFLD rat models.

METHODS

Seven-week-old male Sprague-Dawley rats underwent DMA using IRE after 8 weeks on a high-fat diet. Two weeks post-treatment, duodenal and liver tissues and blood samples were collected. We evaluated differences in the duodenal wall structure, liver lipid deposition, enteroendocrine, claudin, and zonula ocludens-1 in the duodenal mucosa.

RESULTS

DMA using IRE could be safely performed in rats with NAFLD without duodenal bleeding, perforation, or stenosis. The duodenum healed well 2 weeks after DMA and was characterized by slimmer villi, narrower and shallower crypts, and thicker myenterons compared with the sham-control setting. Liver lipid deposition was reduced and serum lipid index parameters were considerably improved in the DMA setting. However, these improvements were independent of food intake and weight loss. In addition, enteroendocrine parameters, such as claudin, and zonula ocludens-1 levels in the duodenal mucosa, differed between the different settings in the DMA group.

CONCLUSION

By altering enteroendocrine and duodenal permeability, simple DMA using IRE ameliorated liver lipid deposition and improved serum lipid parameters in NAFLD rats.

Key Words: Duodenal mucosal ablation; Irreversible electroporation; Non-alcoholic fatty liver disease; Enteroendocrine; Duodenal permeability

Core Tip: This study assessed the feasibility and effectiveness of simple duodenal mucosal ablation (DMA) using irreversible electroporation (IRE) in non-alcoholic fatty liver disease rat model. After DMA using IRE, the duodenal wall structure changed and appeared to return to normal when compared to the sham-control group, while liver lipid deposition reduced and serum lipid parameters considerably improved. Also, changes in the intestinal endocrine system were observed. Therefore, by altering enteroendocrine and duodenal permeability, simple DMA using IRE ameliorated liver lipid deposition in non-alcoholic fatty liver disease rats.

INTRODUCTION

The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing each year, with a global adult prevalence rate of approximately 30% in 2023[1]. Moreover, it is a leading cause of end-stage liver disease as well as primary liver cancer[2,3]. A healthy lifestyle is the most effective therapeutic option for preventing NAFLD onset and progression; however, low compliance limits its efficacy[4]. Further, several methods with different mechanisms of action have been developed to counteract its progression; however, the results remain unsatisfactory[5-7]. Metabolic surgery, an alternative NAFLD therapy, has shown beneficial effects in glycemic control and weight reduction compared with drug therapy alone; however, its short-term and long-term complications cannot be ignored[8,9].

Recently, endoscopic bariatric and metabolic therapies have become safe and effective alternatives for treating NAFLD in clinical practice[10-12]. Duodenal mucosal ablation (DMA) is considered a potential disease-modifying intervention for type 2 diabetes mellitus (T2DM) and NAFLD. Hydrothermal ablation and electroporation therapy are the two main techniques used for DMA in clinical practice, while photodynamic therapy has only been performed in preclinical research[13]. Among these techniques, DMA through electroporation therapy is achieved using irreversible electroporation (IRE), which disrupts cellular homeostasis, leading to cell death without affecting non-cellular components such as blood vessels, nerves, and the extracellular matrix[14,15]. Recently, a groundbreaking study revealed that DMA using IRE is feasible and safe. The study also indicated that combining DMA with glucagon-like peptide-1 (GLP-1) receptor agonists effectively reduces liver lipid deposition in selected patients with T2DM-related NAFLD[12]. However, the specific contribution of DMA using IRE toward metabolic NAFLD alleviation and its potential mechanisms remain unclear. In addition, existing studies on DMA using IRE have been performed in humans or large animals, whereas no study was performed in small animals, especially rodents, which are ideal for mechanistic studies. Therefore, this study aimed to develop a novel DMA method using IRE in rodents, assess its feasibility and efficacy in NAFLD rat models, and determine the effect of DMA using IRE on enteroendocrine and intestinal permeability.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (age, 7 weeks; body weight, 210-230 g) were obtained from the Experimental Animal Center of Xi’an Jiaotong University, a certified management system for the breeding and delivery of rodents devoted to life science research. The animals were housed separately under standardized conditions with a 12/12 hours dark/light cycle, and food and water were freely available. The NAFLD rat model was established using a high-fat diet (Transfat) for 8 weeks. Rat food intake and body weight were recorded daily for each group. Food intake per rat was calculated by subtracting the weight of the remaining feed after 24 hours from the initial weight. All NAFLD rats were randomly categorized into two groups (Figure 1): The DMA (DMA group, n = 6) and non-DMA (sham-control group, n = 6). All animal procedures in this study were approved by the Animal Experiment Ethics Committee of Xi’an Jiaotong University and performed in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (8^th edition, 2011).

Figure 1  Experimental design scheme.

DMA Procedure in NAFLD rats

In this study, the device for DMA using IRE consisted of an ablation catheter and a square-wave pulse generator. The ablation catheter (diameter: 2.5 mm) was custom-made with three electrodes and a temperature sensor at its end, and a square-wave pulse generator (BPG 300, 3ctest, Suzhou, China) generated a burst of electric pulses. The electric field distribution of the ablation catheter during DMA is shown in Figure 2. DMA was performed using the IRE procedure as follows: First, the rat was anesthetized by intraperitoneal administration of pentobarbital sodium (30 mg/kg), and a midline incision was made on the upper abdomen to expose the stomach and duodenum. Second, the ablation catheter was inserted into the stomach, passing it through the pylorus with assistance to reach the duodenum. Third, DMA was conducted using electroporation induced by a 250 V/cm electric field strength (pulse duration: 100 μs, pulse number: 90, and frequency: 1 Hz) (Video 1). After 5 cm in the axial length of the duodenum was ablated, the electrode catheter was withdrawn. Finally, the two-layer abdominal incision was closed with a continuous 3-0 non-absorbable surgical suture. Postoperatively, all rats were placed in a clean cage and buprenorphine (0.05 mg/kg) and cefuroxime (16 mg/kg) were administered intraperitoneally every 12 hours for 3 days. After obtaining blood samples, all rats were sacrificed with artery air-embolism to obtain the liver and duodenum samples 2 weeks after treatment. After weighing the body and liver, the coefficient of the liver was measured and compared between the two groups.

Figure 2 Operation of duodenal mucosal ablation using irreversible electroporation in non-alcoholic fatty liver disease rats. A-C: Ablation catheter; D: Finite element analysis of electrode electric field distribution during operation. Duodenal mucosal ablation was performed using electroporation induced by a 250 V/cm electric field strength (pulse duration: 100 μs, pulse number: 90, and frequency: 1 Hz); E: Rats receiving duodenal mucosal ablation using irreversible electroporation; F and G: Macroscopic observation of the duodenum preoperatively and postoperatively.

Biomedical index tests

Biomedical indexes, including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acids (TBAs), total cholesterol (T-CHO), triacylglycerol (TG), free fatty acids (FFAs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), gastric inhibitory polypeptide (GIP), GLP-1, cholecystokinin (CCK), and lipopolysaccharide (LPS) levels, were measured 2 weeks after DMA.

Histological analysis

All tissue samples were fixed with 4% paraformaldehyde and transferred overnight to phosphate-buffered saline containing 30% sucrose. The samples were embedded in an optimal cutting temperature compound and then sectioned at 10 μm. Hematoxylin and eosin (HE) staining of the liver, duodenum, and ileum tissues was performed, and oil red O staining of the liver tissues was used to compare the lipid core area among the groups. GLP-1, GIP, and CCK expression levels in the duodenum were evaluated using immunofluorescence staining, while claudin-1 expression and zonula occludens-1 (ZO-1) levels were studied using immunohistochemistry.

Statistical analysis

Statistical analyses were completed using SPSS version 20.0 software (IBM, Chicago, IL, United States). Normally distributed measurement data are presented as means ± SD and analyzed using an unpaired, two-tailed t-test. Non-normally distributed data are expressed as medians and analyzed using the Wilcoxon rank-sum test. Statistical significance was set at P < 0.05.

RESULTS

Validation of the DMA using IRE in the NAFLD rat model

DMA via electroporation therapy was successfully performed in all the NAFLD rats in each group, with no postoperative intestinal bleeding or perforation. All rats survived the operation, and food intake and weight changes after DMA were compared between the two groups. The coefficient of the liver values of the rats in the DMA group were higher than those in the control group (3.19 ± 0.04 vs 2.75 ± 0.17, P < 0.05) (Figure 3). At 2 weeks after DMA, the duodenal mucosa appeared to have healed macroscopically and histologically, without the occurrence of ulceration, inflammation, fibrosis, or stenosis. Histologically, the thickness of each duodenum layer was higher in the DMA than in the control group (total thickness: 1478 ± 134 μm vs 1120 ± 86 μm; myenteron, longitudinal lamina: 76 ± 10 μm vs 60 ± 17 μm; circular lamina: 136 ± 13 μm vs 88 ± 15 μm; submucosa thickness: 73 ± 14 μm vs 67 ± 18 μm; mucosa thickness: 1085 ± 93 μm vs 806 ± 102.6 μm; P < 0.05) (Figure 4, Table 1). Furthermore, DMA resulted in significantly narrower and shallower crypts (crypt depth: 220 ± 52 μm vs 236 ± 51 μm; crypt width: 38 ± 6 μm vs 49 ± 5 μm; P < 0.05) (Table 1) and significantly slimmer villi (villus length: 845 ± 74 μm vs 561 ± 104 μm; villus thickness: 133 ± 32 μm vs 200 ± 110 μm; P < 0.05) (Figure 4, Table 1).

Figure 3 Non-alcoholic fatty liver disease rat characteristics between the two groups. A: Body weight; B: Food intake; C: Coefficient of the liver. Data are expressed as the mean ± SD (n = 6). ^aP < 0.05 vs sham-control group. DMA: Duodenal mucosal ablation; COL: Coefficient of the liver.

Figure 4 Histological examination of the duodenal tissue. A: Complete layer of the duodenal wall, sham-control group; B: Measurement scheme of the length (a long dashed black line) and thickness (a short dashed black line) of the villus, sham-control group; C: Measurement scheme of the depth (a long dashed black line) and thickness (a short dashed black line) of the crypt, sham-control group; D: Measurement scheme of the longitudinal (a continuous black line) and circular (a continuous green line) lamina, sham-control group; E: Complete layer of the duodenal wall, duodenal mucosal ablation group; F: Measurement scheme of the length (a long dashed black line) and thickness (a short dashed black line) of the villus, duodenal mucosal ablation group; G: Measurement scheme of the depth (a long dashed black line) and thickness (a short dashed black line) of the crypt, duodenal mucosal ablation group; H: Measurement scheme of the longitudinal (a continuous black line) and circular (a continuous green line) lamina, duodenal mucosal ablation group. DMA: Duodenal mucosal ablation.

Table 1 Effect of duodenal mucosal ablation using irreversible electroporation on the histomorphometrical parameters of the duodenum in non-alcoholic fatty liver disease rats.

Item	DMA group	Sham-control group	P value
Total thickness, μm	1478 ± 134.2	1120 ± 86.47	< 0.05
Myenteron thickness, μm
Longitudinal lamina	75.86 ± 9.7	60.32 ± 17.21	< 0.05
Circular lamina	136.3 ± 13.37	88 ± 14.56	< 0.05
Mucosa thickness, μm	1085 ± 93.26	805.9 ± 102.6	< 0.05
Submucosa thickness, μm	72.81 ± 14.40	67.18 ± 18.29	NS
Villus length, μm	845 ± 73.82	561.3 ± 103.9	< 0.05
Villus thickness, μm	132.7 ± 31.61	199.7 ± 109.5	< 0.05
Crypt depth, μm	219.1 ± 51.57	235.8 ± 50.78	NS
Crypt width, μm	37.56 ± 5.83	48.63 ± 5.24	< 0.05
Villus/crypt ratio	4.016 ± 0.822	2.497 ± 0.721	< 0.05

Data are expressed as the mean ± SD (n = 6). Normally distributed measurement data are presented as means ± SD and analyzed using an unpaired, two-tailed t-test. Non-normally distributed data are expressed as medians and analyzed using the Wilcoxon rank-sum test. DMA: Duodenal mucosal ablation; NS: Not significance.

Effect of DMA using IRE on liver lipid deposition and serum lipid parameters in NAFLD rat

Histologically, hematoxylin and eosin and oil red O staining revealed significant lipid deposition in the rat liver, droplets of different sizes, a disorganized hepatocyte structure, and changes in the sham-control group, which were significantly improved in the DMA group (Figure 5). In addition, serum AST and TBA levels appeared higher in the DMA group, while ALT levels were higher in the sham-control group; however, the differences were not significant (AST: 31.77 ± 13.24 U/L vs 55.52 ± 12.43 U/L, ALT: 21.65 ± 4.35 U/L vs 23.68 ± 5.17 U/L, TBA: 4.32 ± 0.36 μmol/L vs 3.92 ± 0.40 μmol/L) (Figure 5). Serum TG, T-CHO, FFA, HDL-C, and LDL-C levels significantly differed between the two groups. Serum T-CHO and LDL-C were lower in the DMA than in the sham-control group (T-CHO: 2.08 ± 0.15 mmol/L vs 4.39 ± 0.36 mmol/L, LDL-C: 1.05 ± 0.22 mmol/L vs 4.07 ± 0.38 mmol/L, P < 0.01, Figure 5), whereas serum TG, FFA, and HDL-C were higher in the DMA group (TG: 3.26 ± 0.37 mmol/L vs 2.06 ± 0.19 mmol/L, FFA: 1.76 ± 0.10 mmol/L vs 1.22 ± 0.16 mmol/L, HDL-C: 1.03 ± 0.20 mmol/L vs 0.32 ± 0.03 mmol/L, P < 0.05) (Figure 5).

Figure 5 Liver function parameter test, histological examination, and serum lipid parameter test among different groups. A: Serum aspartate aminotransferase levels; B: Alanine aminotransferase; C: Total bile acids; D-G: Histological examination of the liver tissue via hematoxylin and eosin staining and oil red O staining from the sham-control group and from the duodenal mucosal ablation group; H-L: Serum total cholesterol, triacylglycerol, free fatty acid, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels. Data are expressed as the mean ± SD (n = 6). ^aP < 0.05 vs sham-control group, ^bP < 0.01 vs sham-control group. DMA: Duodenal mucosal ablation; AST: Aspartate aminotransferase; ALT: alanine aminotransferase; TBA: Total bile acid; HE: Hematoxylin and eosin; T-CHO: Total cholesterol; TG: Triacylglycerol; FFA: Free fatty acid; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol.

Effect of DMA using IRE on enteroendocrine and intestinal permeability in NAFLD rats

Serum GLP-1, GIP, and CCK of the NAFLD rats in the DMA group were significantly lower than those in the control group (GLP-1: 10.89 ± 0.12 pmol/L vs 11.39 ± 0.10 pmol/L, GIP: 366.70 ± 5.63 pg/mL vs 409.10 ± 3.58 pg/mL, CCK: 258.01 ± 2.52 pg/mL vs 271.70 ± 5.70 pg/mL, P < 0.05). In addition, GLP-1, GIP, and CCK expression levels and fluorescence intensities in the duodenum were significantly increased in the DMA group compared with those in the sham-control group, as assessed via immunofluorescence staining (Figure 6). Regarding intestinal permeability, serum LPS level was significantly lower in the DMA group than in the sham-control group (646.20 ± 9.10 vs 605.40 ± 11.27, P < 0.01, Figure 7). After DMA using IRE treatment, ZO-1 and claudin expression levels in the duodenum were higher in the DMA group than in the sham-control group (Figure 7).

Figure 6 Enteroendocrine test and representative images of duodenal immunofluorescence staining. A: Serum gastric inhibitory polypeptide levels; B: Glucagon-like peptide-1; C: Cholecystokinin; D-I: Immunofluorescence staining of the duodenum from sham-control and duodenal mucosal ablation groups; J-L: Number of gastric inhibitory polypeptide⁺ cells, glucagon-like peptide-1⁺, and cholecystokinin⁺ cells among different groups. ^aP < 0.05 vs sham-control group, ^bP < 0.01 vs sham-control group. DMA: Duodenal mucosal ablation; GIP: Gastric inhibitory polypeptide; GLP-1: Glucagon-like peptide-1; CCK: Cholecystokinin.

Figure 7 Immunohistochemistry staining of the duodenum among different groups. A-H: Immunohistochemistry staining of the duodenum from the sham-control (A-D) and duodenal mucosal ablation (E-H) groups; I-K: Quantitative analysis of zonula occludens-1, claudin and lipopolysaccharide among different groups. ^bP < 0.01 vs sham-control group. ZO-1: Zonula occludens-1; DMA: Duodenal mucosal ablation; LPS: Lipopolysaccharide.

DISCUSSION

Serval studies have indicated that the duodenum plays a critical pathophysiological role in metabolic homeostasis[16,17]. Based on this, multiple therapies, such as metabolic surgery, retrievable duodenal-jejunal bypass liner (DJBL), and DMA[11,16-19], have been developed for metabolic disorder treatment. Metabolic surgery (also known as bariatric surgery) is the most effective therapy for obesity and T2DM; however, its invasiveness and side effects make it unsuitable for all patients[18]. DJBL is an endoscopic technique that could prevent contact of luminal contents with the duodenal mucosa, and it is a potential disease-modifying intervention for T2DM and NAFLD[19-21]. However, the high incidence of complications (gastrointestinal bleeding, hepatic abscesses, and pancreatitis) makes DJBL a less appealing treatment option[22].

DMA is an endoluminal procedure involving hydrothermal ablation or IRE in clinical practice. DMA with hydrothermal ablation, also known as duodenal mucosal resurfacing, requires a submucosal saline injection before each ablation, which is complex and time-consuming. Furthermore, postprocedural gastrointestinal symptoms resulting from the inflammatory response after epithelial necrosis and potential thermal damage to deeper structures should not be ignored[23]. IRE, a non-thermal ablation technique, induces cell apoptosis, necrosis, and immunogenic cell death[14] and has been applied in the treatment of tumors[24], arrhythmia[25], and chronic obstructive pulmonary disease[26]. A recent study revealed that DMA via electroporation therapy combined with semaglutide can replace insulin therapy and improve glycemic control and metabolic health in selected patients with T2DM[12]. However, the specific contributions of DMA, semaglutide, or weight loss toward metabolic disorder recovery are unclear owing to the uncontrolled nature of that study. The mechanism of action of DMA using IRE warrants further investigation. In the current study, we successfully developed a technique for DMA using IRE in rodents and verified its safety and effectiveness in NAFLD rats. To the best of our knowledge, this is the first study to investigate the application of DMA using IRE in small animals. The results showed that IRE can effectively ablate the duodenal mucosa without intestinal bleeding, perforation, or stenosis in small animals. In addition, metabolic parameters and hepatic fat infiltration significantly improved after simple DMA using IRE without the need for other therapies in NAFLD rats fed with a high-fat diet.

Unlike the clinical practice of DMA using IRE, where the ablation electrode is placed in the duodenal lumen under endoscopy, the ablation electrode in this study passed through the pylorus and arrived at the duodenum under guidance after laparotomy. This can occur for several reasons, including the following: (1) The instrument used for clinical DMA via electroporation therapy is the newly developed generation 2 catheter ReCET system (Endogenex, Plymouth, MN, United States), which is not suitable for small animals, especially rodents; and (2) Colonoscopy in rats is relatively risky, more expensive, and invasive, making it unsuitable for DMA experiments in rats.

Regarding the safety and effectiveness of DMA using IRE in rodents, no duodenal bleeding or perforation occurred immediately after ablation. The duodenum healed well, with no evidence of ulceration, inflammation, fibrosis, or stenosis 2 weeks after DMA. Each layer of the duodenal wall changed after ablation, manifesting as slimmer villi, wider and deeper crypts, and thicker myenterons. These changes in the duodenal wall differed from those observed using other DMA methods, such as hydrothermal and photodynamic therapies, where only the mucosal layer was ablated. However, because of the characteristics of the electric field acting on the tissues, every layer of the duodenum was ablated in DMA using IRE. This may have led to intestinal perforation and stenosis; however, no adverse events were observed in this study, mainly because non-cellular components such as the extracellular matrix, blood vessels, and nerves were unaffected during ablation.

Further, lipid deposition in the liver reduced and serum lipid parameters significantly improved 2 weeks after DMA using IRE, consistent with the results of previous clinical studies using hydrothermal therapy[27-30]. However, some differences were observed between our study and previous studies. In previous studies, NAFLD resolution after DMA was usually accompanied by weight loss; however, no changes in the weight and daily food intake of rats after DMA were observed in this study. A possible explanation for this phenomenon is that the postoperative observation time in this study was relatively short, and further studies with longer postoperative observation periods are necessary. However, the decrease in liver lipid deposition and improvement in serum lipid parameters were obvious 2 weeks after DMA, independent of weight loss and food intake. This phenomenon has also been confirmed in a previous study on alleviating NAFLD via duodenal-jejunal bypass[31]. Thus, other mechanisms beyond weight loss underlying metabolic improvement after DMA exist. A systematic review indicated that DMA may alleviate NAFLD by altering intestinal endocrine function or intestinal permeability[32]. In this study, serum GLP-1, GIP, and CCK levels were significantly lower in the DMA group than in the control group. The number of GIP⁺ and CCK⁺ cells in the duodenal wall was different between the groups. In contrast, the number of GLP-1⁺ cells was comparable between the two groups. Serum GLP-1 secreted by L cells has been evaluated in obese rodents fed high-fat and high-sugar diets, which may be a compensatory response to insulin resistance[33]. In our study, rats exhibited lower serum GLP-1 levels 2 weeks after DMA administration, which was accompanied by reduced liver lipid deposition. These results indicate that the insulin resistance of NAFLD rats may reduce after DMA. Moreover, serum GIP and CCK levels, secreted by K and I cells, respectively, were lower in the DMA group, possibly due to decreased duodenal enteroendocrine cells secreting these two hormones. However, the specific contributions of the decreased serum GLP-1, GIP, and CCK levels to the observed metabolic findings remain unclear. Moreover, duodenal permeability improved after DMA using IRE in rats with NAFLD. Studies have indicated that increased chronic low-grade endotoxemia and intestinal permeability are NAFLD and T2DM characteristics[34-39], and improving intestinal permeability could be a treatment option for NAFLD[40-42]. In this study, duodenal permeability improved after DMA using IRE, manifested by a decrease in serum LPS levels and an increase in ZO-1 and claudin expression in the duodenum, accompanied by reduced liver lipid deposition and improved serum lipid parameters. These findings suggest that DMA using IRE may alleviate lipid metabolism disorders through multiple mechanisms, warranting further mechanistic studies.

This study has certain limitations. First, the ablation catheter used in this study was placed in the duodenum after laparotomy, which is an invasive procedure. However, in rodents, the ablation catheter rarely reaches the duodenum without assistance after laparotomy. Non-invasive methods for ablation catheter placement in rodents are needed in future studies. Second, the observation period after DMA was limited to 2 weeks, which may have limited the evaluation of the duration of the positive effect on liver lipid deposition. Notably, our preliminary experiment demonstrated that the effect of DMA using IRE on glycemic control was particularly pronounced 2 weeks post-treatment in T2DM. However, whether repeated DMA using IRE is indispensable for reducing liver lipid deposition and improving serum lipid parameters in NAFLD remains unclear and warrants further study. Third, the relatively small sample size in this study limits the generalizability of the findings and introduces potential selection bias. Therefore, future studies with larger sample sizes are needed to increase statistical power.

CONCLUSION

In summary, DMA using IRE can be safely performed in NAFLD rats. The duodenal wall structure changed and appeared to return to normal when compared to the sham-control group. Liver lipid deposition reduced and serum lipid parameters considerably improved after DMA using IRE. However, these effects were independent of weight loss and food intake. Changes in the intestinal endocrine system were observed, manifested by decreased serum GLP-1, GIP, and CCK levels. However, further studies are necessary to determine the mechanism of action of DMA using IRE in alleviation of metabolic disorders.

ACKNOWLEDGEMENTS

The authors thank Professor Yin Jun from Shanghai Jiaotong University Affiliated Sixth People’s Hospital for his guidance on the experimental design.