Animals

Sixty male 4-week-old C57BL/6J mice (initial body weight: 20.16 ± 1.06 g) were obtained from the Institute of Developmental Biology and Molecular Medicine at Fudan University. Mice were housed in standard-sized cages with ventilation racks, maintained in a pathogen-free, controlled environment at a temperature of 23 ± 1°C and humidity of 60 ± 5%. They followed a 12-h light-dark cycle (from 8 a.m. to 8 p.m.) and had ad libitum access to food and water. The diet consisted of either a standard rodent chow diet or a HFD (D12492) comprising 60% kcal from fat, 20% from protein, and 20% from carbohydrates, primarily in the form of saturated fat. All procedures were approved by the Animal Ethical Committee of Fudan University and followed the guidelines set forth in the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health.

T2D was induced in mice through the administration of a low-dose streptozotocin (STZ) injection following a 5-wk HFD regimen. In the 5th wk of the HFD intervention, mice received intraperitoneal injections of STZ at a dose of 40 mg/kg body weight [ , , ] or an equivalent dose of citrate buffer solution over consecutive three d. Freshly prepared STZ and citrate buffer solution were administered daily. After the STZ injection, fasting blood glucose (FBG) levels were measured using the OneTouch Verio Reflect™ Blood Glucose Monitoring System. This method aligns with Wang et al.’s study, which characterizes T2D by elevated blood glucose levels and reduced insulin sensitivity, as confirmed through insulin tolerance testing (ITT) [ ].

Mice were divided into six groups (n = 10 per group), with an equal number of mice in each group: Control mice (C-Sham), control mice with sham LIPUS exposure (C-LIPUS), T2D mice with sham LIPUS exposure (T2D-Sham), T2D groups treated with LIPUS at different spatial average-temporal-average intensity ( I _sata ) , that is 60 mW/cm² (T2D-L-60), 80 mW/cm² (T2D-L-80), and 100 mW/cm² (T2D-L-100). During the group allocation process, body weight and blood glucose levels were carefully balanced across all groups.

LIPUS treatment

The mice groups received LIPUS stimulation for 20 mins per session, five days a wk, over a period of six wks with a frequency of 1 MHz, a duty cycle of 20%, and I _sata levels of 60 mW/cm², 80 mW/cm², and 100 mW/cm² respectively. The C-LIPUS group underwent a similar procedure with ultrasound that was not activated. The LIPUS was generated using a custom ultrasonic therapy device ( Fig. 1a ) developed by the Intelligent Medical Ultrasound Lab at Fudan University (Shanghai, China) [ , ]. Ultrasound parameters, including duty cycle, frequency, PRF and treatment duration, were manually adjusted to specific levels (duty cycle of 20%, frequency of 1.5 MHz, PRF of 1.0 kHz and daily exposure duration of 20 mins). Intensity levels were modulated during the exposure and calibrated at the beginning and throughout the experiment using a high-resolution (1 mW), fast-response (<3 s) ultrasound power meter (UPM-DT-1000 PA, Ohmic Instruments, MO, USA). Mice were depilated and then continuously anesthetized using continuous inhalation of isoflurane to maintain a horizontal prone position. The targeted region for LIPUS therapy BAT in mice, located in the interscapular back neck area [ ], was exposed to a 25-mm diameter transducer (Suzhou, China), with ultrasonic coupling gel applied between the skin and the circular transducer to ensure optimal contact and transmission of ultrasound waves. The entire experimental process is schematically illustrated in the timeline depicted in Figure 1b .

(a) Experimental schematic diagram. A schematic diagram of the mouse receiving LIPUS treatment in a prone horizontal position, with the treatment area focused on the scapular and neck regions targeting BAT (b) Whole experiment timeline.

Glucose and insulin tolerance test

To assess glucose uptake and insulin resistance, we performed intraperitoneal GTT and intraperitoneal ITT five wks following LIPUS exposure. Mice underwent a 12-h fasting period before the intraperitoneal GTT with an administration of 1 g/kg glucose. The ITT with an administration of 0.5 unit/kg insulin was conducted, requiring a 6-h fasting period from 9 am to 3 pm for the mice. Blood samples were collected through a small incision in the tail, and blood glucose concentrations were measured at intervals of 30, 60, 90, and 120 minutes after the intraperitoneal injections. Results were subsequently determined using the area under the curve (AUC) method.

NMR of body composition

To assess the impact of LIPUS on the body composition of the mice, measurements were taken for fat mass and free liquid composition using Time Domain NMR technology with the Bruker LF50H TD-NMR minispecTM. The analyzer is 70 cm wide and 110 cm deep. Measurements are radiation-free (no special shielding is needed in requirement), rapid, completely silent, and offer a highly accurate and user-friendly method for assessing fat content in mice without anesthesia. Raw NMR data were processed using Bruker software to calculate body composition parameters, including fat mass and lean mass, among others.

Metabolic cage

To evaluate the impact of LIPUS on metabolic function, a 72-h metabolic cage test was conducted after 4 wks of LIPUS exposure. The Promethion Metabolic Cage System from Sable (Nevada, USA) was utilized to monitor individual mice’s oxygen consumption, carbon dioxide production, water intake, food intake and activity over 72 h. The metabolic exchange rate is determined by the ratio of carbon dioxide production to oxygen consumption.

Serum biochemical analysis

Mouse blood extracted at sacrifice was analyzed for total cholesterol (T-Cho) and triglyceride (TG), tumor necrosis factor-alpha (TNF-α) and Interleukin-1β (IL-1β). Following blood collection, samples were promptly centrifuged at 3500 rpm for 15 minutes at 4°C to separate the serum. The serum was then stored at -80°C until further biochemical analysis. Serum T-cho and TG in serum were measured following the manufacturer’s instructions (A111-1-1, A110-1-1, Nanjing Jiancheng Bioengineering Institute, China). Serum insulin (90080, Ultra-Sensitive, USA), TNF-α (MEC1003, Anogen, Canada), and IL-1β (MEC1010, Anogen, Canada) were quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer instructions.

Histologic analysis

After sacrifice, two sides of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (sWAT), and BAT from the interscapular and back neck areas were excised from the mice and weighed. BAT was divided into three pieces, and sWAT into two pieces. All of two pieces of SWAT, two pieces of BAT and eWAT were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned for H&E staining and UCP1 immunohistochemical (IHC) staining. The stained slides were analyzed using an optical microscope (Nikon Eclipse E100), and standard scanning images were generated. CaseViewer software (Version 2.4.0, 3D Histech, Budapest, Hungary) was utilized to review the scanned images, and ImageJ software (Version 1.53n, National Institutes of Health, USA) was employed to measure the diameter and area of adipocytes as well as the area of UCP1 expression.

Real-time quantitative PCR (qRT-PCR)

The last piece of BAT was snap-frozen in liquid nitrogen for PCR analysis. Then, the tissue was utilized for total RNA extraction, followed by reverse transcription to generate cDNA. Subsequently, qRT-PCR analysis was conducted to quantify the expression of proinflammatory cytokines, including TNF-α and IL-1β. Total RNA was isolated from adipose tissue samples with TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. The RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from 1 µg of total RNA using the RR037A kit (Takara) in a total volume of 20 µL. qRT-PCR was carried out using the SYBR Green PCR Master Mix (TaKaRa, China) on an ABI 7900HT sequence detection system. The reaction mixture consisted of 10 µL SYBR Green Master Mix, 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 2 µL cDNA template and 6 µL nuclease-free water, for a final volume of 20 µL. The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 mins, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed using the comparative Ct (ΔΔCt) method with GAPDH as the internal control. The primers used for qRT-PCR are listed in Table 1 .

Statical analysis

All results were presented as mean ± standard error of the mean (SEM). Data were analyzed using GraphPad Prism software (Version 8.3.0, San Diego, USA). Statistical comparisons between two groups were assessed using a two-taile d Student’s t-test, while differences among multiple groups were assessed using two-way analysis of variance (ANOVA) followed by posthoc Holm-Sidak multiple comparison tests.

Establishment of T2D mice model

Following STZ and citrate buffer injection in the 5th wk of HFD, confirming the establishment of the T2D mice model involved checking the FBG levels, which exhibited a significant elevation compared to the chow-diet-fed mice ( p < 0.0001) ( Fig. 2a ). Simultaneously, there was a notable increase in the body weights of HFD-fed mice, reaching 34.55 ± 2.1 g, in contrast to the chow-diet-fed ( p < 0.0001) at 27.8 ± 1.2g ( Fig. 2b ). According to the protocol, mice with blood glucose levels exceeding 180 mg/dl (10.3 mmol/L) [ ], were selected to represent the established T2D model. When the mice were divided into six groups, weights were carefully balanced between the T2D-Sham group, T2D-L-60, T2D-L-80 and T2D-L-100 groups. These mice were designated as T2D mice and subsequently used as recipients for the LIPUS treatment experiments.

Establishment of the T2D mouse model (a) FBG levels in the control group mice and T2D model mice; (b) Weight comparison between the control group mice and T2D model mice.

LIPUS alleviating insulin resistance in T2D mice

After a 4-wk exposure to LIPUS, GTT and ITT were conducted to assess insulin resistance across all experimental groups. In control groups, LIPUS treatment showed no significant effects in the C-LIPUS group ( p = 0.1229) ( Fig. 3a –f) compared to the C-Sham group. In comparison with C-Sham group, the T2D-Sham group exhibited substantial impairment in glucose tolerance ( p < 0.0001), as evidenced by the intraperitoneal GTT ( Fig. 3a ) and the corresponding AUC ( Fig. 3b ). Additionally, reduced insulin sensitivity was observed in the T2D-Sham group mice ( p < 0.0001), as indicated by the ITT ( Fig. 3d ) and the AUC of the ITT ( Fig. 3e ).

LIPUS improves glucose tolerance and insulin sensitivity in T2D mice (a) Blood Glucose Levels during GTT; (b) AUC of GTT; (c) Serum insulin (ng/ml); (d) Blood glucose levels during ITT; (e) AUC of ITT; (f) Homeostatic model assessment of insulin resistance (HOMA-IR).

Overall, mice treated with LIPUS at intensities of 60, 80 and 100 mW/cm² demonstrated enhanced glucose tolerance or improved insulin resistance as opposed to the T2D-Sham group ( p < 0.0001, p < 0.0001 and p = 0.0006, respectively), as evidenced by both GTT and ITT outcomes ( Fig. 3a , 3b , 3d , 3e ). Notably, the T2D-L-60 and T2D-L-80 groups exhibited superior tolerance in GTT compared to the T2D-L-100 group ( p = 0.0287 and p = 0.0272) ( Fig. 3a , 3b ). In addition, the T2D-L-80 group demonstrated significantly enhanced insulin tolerance compared to the other two treatment groups T2D-L-60 ( p = 0.0302) and T2D-L-100 ( p = 0.0136) ( Fig. 3d , 3e ). Moreover, the serum insulin levels of the T2D-L-100 group exhibited lower insulin levels compared to the T2D-L-60 ( p = 0.0282) and exhibited lower trends compared to T2D-L-80 ( p = 0.2073) groups ( Fig. 3c ). Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) analyses unveiled that all the treatment groups exhibited lower insulin resistance levels compared to the T2D-Sham group ( p < 0.0001) ( Fig. 3f ). Among the treatment groups, the T2D-L-80 group demonstrated trend toward reduced insulin resistance levels compared to the T2D-L-60 group ( p = 0.1492) and was significantly lower than the T2D-L-100 group ( p = 0.0087) ( Fig. 3c , 3f ).

LIPUS reduces body weight, fat content and lipid levels

NMR body composition analysis and blood lipid levels revealed that the C-Sham and C-LIPUS groups exhibited no significant differences in serum TG and T-Cho concentrations ( Fig. 4c , 4d ). Conversely, the T2D-Sham group mice demonstrated significantly higher obesity and blood lipid levels as opposed to the control groups ( p < 0.0001) ( Fig. 4c , d). Overall, the T2D-L-60 (mean weight = 29.8364), T2D-L-80 (mean weight = 29.47), and T2D-L-100 (mean weight = 29.8) groups showed weight loss compared to the T2D-Sham group ( p = 0.0031, p = 0.0038, and p = 0.0024) ( Fig. 4 a) and a decrease in fat content ( p = 0.0002, p = 0.0002, and p < 0.0001) ( Fig. 4b ). Additionally, mice treated with LIPUS exhibited a significant reduction in fat content in both sWAT ( p < 0.0001) and eWAT ( p < 0.0001) ( Fig. 4f , 4g ) compared to the untreated group. T2D-L-80 and T2D-L-100 groups displayed showed a trend toward reduced serum TG concentration compared to the T2D-Sham group mice ( p = 0.0900 and p = 0.1797 respectively) ( Fig. 4c ). Additionally, compared to the T2D-Sham group, T2D-L-60 and T2D-L-100 groups exhibited significant smaller BAT mass ( p = 0.0466 and p = 0.0213, respectively), T2D-L-80 group displayed a tendency to lower BAT mass ( p = 0.3150) ( Fig. 4e ). All treatment groups showed a trend toward reduced serum T-Cho concentration ( Fig. 4d ) ( p = 0.6724, p = 0.5445 and p = 0.2219, respectively) ( Fig. 4d , 4e ). Notably, the T2D-L-80 group mice revealed a tendency towards lower body weight ( Fig. 4a ) but higher fat mass ( Fig. 4b ) with a greater proportion of BAT content relative to WAT content relative to the T2D-L-60 ( p = 0.6319, p = 0.4972) and T2D-L-100 groups ( p = 0.5576, p = 0.3442) ( Fig. 4e , 4f ). Furthermore, we did not observe any significant difference in food intake between T2D mice and those receiving LIPUS treatment ( Fig. 4h ).

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