The study protocol was approved by the Institutional Review Board of the Chengyang People's Hospital in Qingdao (China; approval no. CYQRMYY2019-09-11) and performed following the 1964 Declaration of Helsinki and its later amendments. All study participants provided written informed consent. A total of 39 patients (26 female and 13 male) aged 20-75 years who were overweight or obese (BMI, 27-40 kg/m²) and diagnosed with IGT from October 2019 to September 2021 in Chengyang People's Hospital were included in the present study. Glucose tolerance status was defined using an oral glucose tolerance test (OGTT) according to the World Health Organization criteria (20). The exclusion criteria were as follows: i) Type 1 or 2 diabetes; ii) lower limb ischemia; iii) treatment with hypoglycemic drugs or angiotensin-converting enzyme inhibitor/angiotensin II receptor antagonist during the trial; iv) chronic disease or health conditions; v) acute illness (such as infection) and vi) pregnancy or lactation.

The patient baseline clinical characteristics, including age, sex, height, weight, BMI, waist circumference (WC), hip circumference (HC), systolic blood pressure (SBP) and diastolic blood pressure (DBP), were recorded.

The study period was October 2009 to September 2021. Participants were randomized to receive either liraglutide (n=17) or lifestyle interventions (n=22) for 6 months. Liraglutide was injected subcutaneously daily before breakfast. The dose was escalated weekly from 0.6 to 1.2 mg (increments, 0.1 mg per week). Lifestyle interventions included dietary intervention and physical activity. The dietary intervention involved advice and counseling to develop an individual plan for behavioral change aiming to achieve the following: Total dietary energy intake >50% from carbohydrates; decreased total and saturated fat intake with <30% total dietary energy from fat intake; increased fiber intake and weight loss to achieve BMI <25 kg/m² (21,22). The physical activity intervention was designed to encourage participation in increased physical activity equivalent to 30 min of moderate aerobic physical activity/day. Both participants and study administrators (physicians, nurses, dietitians and coordinators) were blinded to the treatment assignment.

Following 14 h of overnight fasting, all subjects were admitted to the Chengyang People's Hospital in Qingdao (China) between 8:00 and 9:00 a.m. After resting for 30 min, venous blood (2 ml) was drawn to measure fasting glucose and INS levels. A 75-g oral glucose load was administered over a 1 min period. Blood draws for glucose and analysis of INS levels were performed 60 and 120 min after administration of the glucose load. Serum INS was measured using the Mercodia Insulin ELISA kit (cat. no. 10-1113-01; Mercodia AB) (23).

Blood samples were collected after 14 h overnight fasting at baseline and after 4 months of each treatment. Serum levels of triglyceride (TG) and total cholesterol (TC) were measured using an enzymatic method (Roche Diagnostics). The phosphotungstic acid-Mg²⁺ method was used to determine high-density lipoprotein (HDL) concentrations. Low-density lipoprotein (LDL) was estimated in samples with a triglyceride level <400 mg/dl, using the modified Friedewald formula (24).

The glycosylated hemoglobin (HbA1c) levels were measured through boronate-affinity high-performance liquid chromatography (Premier Hb9210™; Trinity Biotech, Inc.). The analytical column contains aminophenylboronic acid bonded to a porous polymer support (gel) and pumps transfer reagents and patient samples through the analytical column. Modules of the instruments are as follows: SPD 20A UV detector, DGU-20A5 degasser, SIL-20A HT autosampler, LC-20AT pump (liquid chromatograph) and CTO-IOAS column oven. Briefly, to a 5 µl sample, a 1,250 µl hemolysis reagent was added and the mixture was left for 30 min at 37˚C. The precipitated protein was removed by centrifugation at 10,000 x g at 4˚C for 2 min. A total of 20 µl of the supernatant was injected into the chromatographic system. Separation of HbA1c was achieved with a 35x4.6 mm cation exchanger column (ImmuChrom GmbH) with a particle size of 3 µm at a flow rate of 1.5 ml/min. The areas of peaks detected by UV detector (415 nm) were used for quantification. HbA1c levels were expressed as %. To minimize inter-batch analytical variation, all samples from any given volunteer were assayed in a single batch. Each sample from one subject was assayed in duplicate, using the analytical system in accordance with the manufacturer's instructions. The same lots of calibrators, reagent lot and quality-control materials were used throughout, and analyses were performed by a single analyst.

C-reactive protein (CRP) levels were measured using the immunoturbidimetric method (CRPL3 assay; Roche Diagnostics) on a Cobas 702 module (Roche Diagnostics). TNF-α (cat. no. YSRIBIO-3736), IL-1β (cat. no. YSRIBIO-3292), IL-2α (cat. no. YSRIBIO-4666) and IL-6 (cat. no. YSRIBIO-4610) levels were measured using ELISA kits obtained from Shanghai Yansheng Biochemical Reagents Co., Ltd. The ELISA procedure was performed according to the manufacturer' instructions. White blood cell (WBC) count analysis was performed using using the Sysmex XE 2100 automated hematology system (Sysmex Corporation) (25).

B-mode real-time ultrasound was performed at baseline and after 4 months of treatment to evaluate arterial wall thickness in the carotid arteries as a surrogate marker of subclinical atherosclerosis (26). All examinations were performed by a single examiner in a blinded manner using the same ultrasound machine (Acuson Antares™ ultrasound system, premium edition; Siemens Healthineers), without access to previous scans when follow-up studies were performed. The ultrasound examination was performed in a standardized manner and specific sonographic images were obtained for comparison.

Patients were examined in the supine position and each carotid wall or segment was evaluated to identify IMT, as previously reported (27). Each scan of the common carotid artery began just above the clavicle and the transducer was moved to the carotid bifurcation and along the internal carotid artery. In total, three segments were identified and measured in anterior and posterior planes on each side: i) Distal 1.0 cm of the common carotid artery proximal to the bifurcation; ii) the bifurcation and iii) proximal 1.0 cm of the internal carotid artery. At each of these sites, IMT was determined, defined as the distance between the echogenic line representing the intimal blood interface and the outer echogenic line representing the adventitial junction. IMT <0.9 mm was defined as normal; 0.9 mm≤ IMT <1.3 mm was defined as intimal medial thickness; and IMT ≥1.3 mm was defined as plaque. In addition, electrocardiogram revealed the general condition of the coronary arteries. ST segment or T wave change on electrocardiogram was considered as myocardial ischemia.

The primary endpoint was occurrence of atherosclerosis in macrovasculature, as well as in peripheral and visceral vessels. Patients were followed up once every 3 months, with a total follow-up duration of 6 months. The patient clinical profile and laboratory measurements were recorded at each visit. The side effects during the intervention were monitored to assess the tolerance and safety of each treatment.

Statistical analysis was performed using SPSS version 22.0 (IBM Corp.). The normality of distribution of the variables data was assessed using Shapiro-Wilk test. Data normally distributed are presented as mean ± standard deviation and tested using Student's t test. If the data were skewed, the Wilcoxon rank sum test was used to assess differences in changes before and after treatment between the two groups. Categorical data was compared using χ² test or Fisher's exact probability method. Kaplan-Meier survival analysis and a log-rank test were used to compare the risk of vascular disease following treatment between the two groups. A two-tailed P<0.05 was considered to indicate a statistically significant difference.

Individuals randomized to receive liraglutide or lifestyle intervention had similar baseline characteristics including sex (P=0.819), age (P=0.322), BMI (P=0.596), WC (P=0.734), HC (P=0.292), DBP (P=0.795) and SBP (P=0.547) (Table I). BMI, WC and HC after liraglutide treatment were lower than those after lifestyle intervention (all P<0.001). DBP and SBP levels did not show any statistically significant difference between the treatment arms (all P>0.05).

Treatment-associated side effects are shown in Table SI. The most common side effect of liraglutide was nausea (n=4; 23.5%). There was no significant difference in side effects between liraglutide treatment and lifestyle intervention.

Liraglutide treatment could induce a greater reduction in HbA1c compared with lifestyle intervention (P<0.001; Table II). Similar trends were also observed for fasting blood glucose, fasting INS, 2 h postprandial blood glucose and 2 h postprandial INS between liraglutide treatment and lifestyle intervention (all P<0.001; Table II).

Liraglutide treatment significantly decreased TC and LDL levels compared with lifestyle intervention (all P<0.001; Table III). However, no changes in TG and HDL were observed.

Liraglutide treatment significantly decreased levels of WBC, CRP, TNF-α, IL-1β, IL-2α and IL-6 compared with lifestyle intervention (all P<0.001; Table IV).

The levels of CIMT decreased significantly in patients treated with liraglutide compared with lifestyle intervention (P<0.001; Table V). Although the lifestyle intervention group exhibited a higher incidence of atherosclerosis in coronary and peripheral arteries compared with that in the liraglutide treatment group, no significant difference was observed (all P>0.05; Table SII). Based on the Kaplan-Meier analysis, the incidence of vascular disease in the liraglutide group was significantly lower than that in the lifestyle intervention group (log-rank test P=0.041; Fig. 1).