Study design

This trial is registered in Acupuncture-Moxibustion Clinical Trial Registry, (AMCTR, http://www.acmctr.org/, No. AMCTR-IOR-20000341). It follows the principles of the Declaration of Helsinki and the Good Clinical Practice Guideline. This study was reported following the Consolidated Standards of Reporting Trials (CONSORT) [24] and STandards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) [25] reporting guidelines. It has been reviewed and approved by the medical ethical committee of Zhongshan Hospital, Fudan University (Approved No. B2019-310R). Before randomization, all participants were requested to sign a written informed consent to decide whether they were willing to participate in this trial. They were informed of the details of the study and all the benefits and risks of participating in this trial.

Participants

All participants were diagnosed with T2DM, and referred by a doctor. Diagnosis of the T2DM was performed as presenting 1 or more of the following: (1) fasting plasma glucose (FPG) ≥ 7.0 mmol/L, (2) 2-h plasma glucose (2-h PG) value during a 75-g oral glucose tolerance test (OGTT) ≥ 11.1 mmol/L, (3) random plasma glucose ≥ 11.1 mmol/L. Referring to the Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults [26], overweight or obesity was defined as body mass index (BMI) ≥ 24 kg/m2 or ≥ 28 kg/m2. Other inclusion criteria were age, from 18 to 70 years old, and voluntary informed consent. The exclusion criteria included pregnancy and lactation; long-term courses of hormone drugs or weight loss supplements, severe organic diseases, such as advanced tumor, cardiac, hepatic, renal, or mental diseases; and unfit to receive treatment of acupuncture. After eligibility, informed consent was obtained from the participants and then confirmed.

The sample size calculation was based on the primary outcome. According to our pilot study, the mean reduction of body weight by a combination of exercise and acupuncture was 4.2 kg, with a standard deviation (SD) of 1.5 kg. To yield an 80%-statistical power with a two-sided significance level of 0.05, 36 participants per group were needed. Furthermore, assuming a dropout rate of 10%, meanwhile, referring to the requirement from higher authority (every group contains ≥ 50 participants), a total of 120 participants were determined.

Randomization and blindings

Qualified participants were randomly assigned by a computer-generated list to the acupuncture or control (sham acupuncture) group according to a ratio of 1:1. An independent mathematician used R 4.0 to generate a randomized sequence according to the order of participant enrollment, and a third party concealed the allocation sequence in sealed opaque envelopes. The acupuncturist will be made aware of group assignments prior to treatment. Throughout the study, participants, data collection staff, and data analysts will remain blinded to the treatment assignments. Additionally, participants will receive their treatments in separate rooms or at different times to prevent communication between them, and the acupuncturist will not disclose any information about the treatment programs to the participants. In the data summary stage, the three separation principles of researcher, operator, and statistician will be implemented. The control group followed a CR diet and engaged in exercise and sham acupuncture, while the acupuncture group received the diet guideline and engaged in exercise and acupuncture interventions.

InterventionDiet protocol

The CR intervention was designed to reduce body weight via decreasing dietary consumption, meanwhile improving eating habits. All participants were prescribed a balanced diet that provided an energy deficit of 500 kcal per day, with a macronutrient distribution within the range of 45–60 energy percent (E%) carbohydrate, 15–20 E% protein, and 20–35 E% fat (< 7 E% saturated fat). The dietitian would provide individualized recommendations and recipes based on the qualitative evaluation of food diaries daily.

Exercise protocol

The exercise intervention was designed to increase energy expenditure and reduce lean mass loss to improve body composition via combining aerobic and resistance exercises [27, 28]. All participants take exercise five times weekly, including three times aerobic and two times resistance exercise. The exercise was approximately 30–40 min, followed by a sequence of static stretching exercises (~ 20 min) designed to improve flexibility. The types of aerobic exercise included brisk walking, jogging, cycling, swimming, and Taiji. The resistance training consists of a sequence exercise for upper-extremity and lower-extremity exercises, including shoulder presses, push-ups, dumbbell front raise, plank, squats, and glute bridge. Participants exercised at approximately 65% of their peak heart rate, which was gradually increased to 70 to 85% to reach the criteria of moderate-intensity exercise. The exercise is conducted under the distance instruction and supervision of a fitness instructor.

Acupuncture protocol

According to the theory of channels of Traditional Chinese Medicine, the following acupoints of Foot Yang Ming stomach channel were chosen refer to previous systematic reviews and consultations with specialists [23, 29]: Renying (ST9, level with the tip of and 1.5 cun lateral to the laryngeal prominence), Shuitu (ST10, 1 cun down from ST9, on the anterior border of sternocleidomastoideus muscle), Liangmen (ST21, 4 cun above the midline of the umbilicus, 2 cun lateral to the anterior midline of the abdomen), Tianshu (ST25, 2 cun lateral to the midline of the umbilicus), Guilai (ST29, 4 cun below the midline of the umbilicus, 2 cun lateral to the anterior midline of the abdomen), Zusanli (ST36, 3 cun below the lateral depression of patellar ligament, one finger width lateral from the anterior crest of the tibia), Fenglung (ST40, 8 cun superior to the tip of the external malleolus, two finger width lateral from the anterior crest of the tibia), Neiting (ST44, Proximal to the web margin between the 2nd and 3rd toes, at the junction posterior to the toe web of the red and white skin).

The acupuncture was completed by a professional acupuncturist with a Chinese medicine practitioner license from the Ministry of Health of the People’s Republic of China. In the authentic acupuncture group, each point was penetrated with disposable acupuncture needles (45 mm in length and 0.3 mm in diameter, Suzhou Medical Appliance Factory, Jiangsu Province, China) approximately 30 to 40 mm into the skin. The needles in ST25, ST29, ST36, and ST40 were connected to an electro-stimulator (G6805-2 A, Shanghai Huayi Medicinal Instruments Co., Ltd, Shanghai, China), producing electrical stimulation with a continuous 1 Hz wave for 10 min. In the sham acupuncture group, the needles were penetrated only 2–3 mm into the skin, and the needles in ST25, ST29, ST36, and ST40 were connected to the electro-stimulator without electrical stimulation. The needles were left for 30 min. The procedure was carried out once every 2 days, 15 times in total.

Outcomes measurementPrimary outcome measure

The primary outcome was the change in body weight after treatment.

Secondary outcome measure

Secondary outcomes included the change in BMI, waist-to-hip ratio (WHR), FPG, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and body fat content. The complete body measurements and scheduled laboratory tests were conducted before and after the intervention.

rs-fMRI outcome measures

The MRI experiment was performed using a 3.0 Tesla MR device (Discovery MR750, GE Healthcare, Wisconsin, USA), which is equipped with an 8-channel phased array head coil. The rs-fMRI data were acquired using an echo-planner imaging sequence with the following parameters: repetition time (TR) = 986 ms; echo time (TE) = 30 ms; flip angle (FA) = 52°; voxel size = 2.2 × 2.2 mm; field of view (FOV) = 220 × 220 mm2; slice thickness = 2.5 mm, 78 axial slice. Subsequently, high-resolution 3-Dimensional T1-weighted images were acquired using a whole-brain sagittal spoiled gradient echo (TR = 8.26 ms; TE = 3.24 ms; FA = 8°; voxel size = 0.8 × 0.8 × 0.8 mm; FOV = 256 × 256 mm, and 256 sagittal slices). During the MRI scan, participants were instructed to close their eyes but to remain awake.

MRI-PDFF outcome measures

The acquisition was performed during a single breath-hold of 21 s. PDFF, and water fraction, were acquired automatically via iterative decomposition of water and fat with echo asymmetry and least squares estimation quantification. The parameters were listed as follows: TR = 6.4 ms, TE = Min Full, FA = 3°, matrix size = 160 × 160; FOV = 35 × 24 cm; bandwidth = 111.11 kHz, 24 axial slices with 10 mm slice gap. Quantitative analysis of PDFF was completed by a senior radiologist blinded to the subjects’ information. For the quantification of fat content, three regions of interest (ROI) with 150 mm2 in the area were placed at different locations in every interested organ, including the liver, pancreas, and psoas, avoiding the adjacent structures and the main vessel, and the average of the three ROIs was considered as the result of MRI-PDFF.

Data and statistical analysisMRI data analysis

The MRI data were pre-processed using the DPABI toolbox v8.0 (www.rfmri.org) on the Matlab R2019a platform. This process mainly contained 6 steps: (1) DICOM format conversion, (2) removing the first ten scan volumes, (3) section timing correction, (4) correction of head movement, (5) spatial standardization, and (6) spatial smoothing. The images were spatially normalized into the standard Montreal Neurological Institute (MNI) template with a resampling voxel size of 3 × 3 × 3 mm3. Temporal bandpass filtrating (0.01–0.1 Hz) was performed to reduce the effect of low-frequency drifts and high-frequency noise.

Analysis of fractional amplitude of low-frequency fluctuation (fALFF) and regional homogeneity (ReHo) analysis was performed using the DPARSF software in the DPABI toolbox. After spatial standardization, the linear tendency of the preprocessed data was removed by the linear regression method. To obtain individual fALFF values, the fast Fourier transform of each voxel signal of the time series in the whole brain image was performed and converted into the square root of the frequency domain power spectrum. In this study, fALFF was obtained as the division of the average square root of the power spectrum at each voxel across 0.01–0.1 Hz by the whole frequency range observed in the signal. Individual ReHo maps were generated by calculating Kendall’s coefficient of concordance (KCC) of the time series of every 27 neighboring voxels. Then the ReHo value was divided by the global mean KCC value in each subject for further statistical analysis. 2 × 2 repeated-measures ANOVA analysis of variance with intervention (control versus acupuncture) and time point (baseline versus follow-up) was performed to assess between-group differently of rs-fMRI activity. The regions with significant changes in the fALFF and ReHo values were considered as uncorrected p < 0.005 with cluster-extent ≥ 10 voxels.

Clinical data analysis

All clinical statistical analyses were performed in R 4.0. The primary outcome, change in body weight, was evaluated by repeated-measures analysis of variance. Baseline differences between groups and other outcome variables were assessed by Student’s t-test, Mann–Whitney U-test, or Chi-square test according to the data characteristics. The paired t-test between before and after interventions was performed with a paired samples t-test or Wilcoxon signed-rank test. Data were presented in mean values with an SD or as a number with a percentage. Significance was defined as a two-tailed value of 0.05.