Adult wild-type AB zebrafish (1:1 male/female ratio, 6–8-month-old with body weight ranging from 0.29 g to 0.43 g, 100 per group) were purchased from the Wuhan Zebrafish Resource Center (Wuhan, Hubei Province, China). They were initially maintained in a five-tier, single-drain aquatic system (Shanghai Haisheng Marine Biological Equipment Co., Ltd., Shanghai, China) under a controlled 14 h light/10 h dark cycle, with a stable temperature of 28.5 $\pm$ 1 °C [21]. Zebrafish were divided into three groups: control, T2DM, and T2DM+exercise (T2DM+ST). The control group received a basal diet (fairy shrimp), while the T2DM and T2DM+ST groups were fed a high-sugar fairy shrimp diet twice daily (0.1 g per 10 fish) [22].

Unfertilized fairy shrimp (10 g, fairy shrimp were purchased from FengNian Aquaculture Co., Ltd., Tianjin, China) were incubated in a 2 L container with 20 g of sea salt (FengNian Aquaculture Co., Ltd., Tianjin, China) and 1.5 L of pure water for 16 h. After hatching, the fairy shrimp were immersed in a 20% D-glucose (#M18078-500G, Meryer, Shanghai, China) solution for 4 h (omitted for the control group), rinsed with ddH₂O, flash-frozen in liquid nitrogen, and subsequently freeze-dried for 36–48 h using a vacuum freeze-drying machine (SCIENTZ-12N, Zhejiang Xinzhi Biotechnology Co., Ltd., Ningbo, Zhejiang Province, China). This provided the basic feed (fairy shrimp) for the control group, and the high-sugar feed (high-sugar fairy shrimp) for the T2DM and T2DM+ST groups.

The training device was adapted from previous experiments [23] and configured to facilitate swimming training (ST) at a maximum flow rate of 10 cm/s for 30 minutes daily over 20 days. Training sessions were consistently scheduled at approximately 2:00 PM. Zebrafish were trained simultaneously across three slots (Fig. 2). The training field was a transparent pipe (Acrylic material) with a diameter of 25 mm, length of 15 cm, and thickness of 3 mm.

Fig. 2.

Zebrafish aerobic exercise equipment. The arrow indicates the direction of water flow. (A) Diagram of the device design. (B) Photograph of the actual device.

A custom-designed trapezoidal transparent fish tank (dimensions: 28 cm upper, 22 cm lower, 15 cm height, 7 cm width) filled to a height of 15 cm with water was utilized. A warm white LED light panel was positioned behind the tank to provide consistent illumination (50 lux) [24]. A digital camera (resolution ratio: 1920 $\times$ 1080) interfaced with a computer was placed in front of the tank to capture the movement trajectory of the zebrafish. Behavioral assessments included the NTT and the shallow water test, using the same set of zebrafish. For the NTT, 10 fish were individually tested, with each fish tested three times in parallel for 6 minutes each time (Supplementary Video 1).

The shoal test was conducted in a transparent acrylic tank measuring 20 $\times$ 20 cm and filled with water to a depth of 2 cm. A warm white LED light plate (Jiaxing Yinjing Electric Co., Ltd., Jiaxing, Zhejiang Province, China) was positioned beneath the tank, and zebrafish behavior recorded with a digital camera (resolution ratio: 1920 $\times$ 1080) placed above the sound stage. The other end of the camera was connected to a computer running Fish Track software (Fish Track, version 2.0 copyright©2019, Shanghai Xinrunke Information Technology Co., Ltd., Shanghai, China). Individual zebrafish were accurately identified through static background analysis and gray threshold, and their movement trajectory was tracked (Supplementary Video 2) [25]. The shoal test was carried out with 10 fish at the same time, with a test time of 6 min.

Weight (g) and body length (cm) were measured using a digital balance (Sartorius, Gogentin, Germany) and an electronic vernier caliper (Deqing Shengtai Core electronic Technology Co., Ltd., Huzhou, Zhejiang Province, China), respectively. The CF was calculated using the formula: CF = (W/L³) $\times$ 100, where W is the weight in g and L is the length in cm [26]. A controlled environment was created by adding crushed ice to a container filled with water, ensuring the water level reached half the depth of the ice, thereby maintaining a consistent temperature range. Ten zebrafish were randomly selected and anesthetized via ice-bath immersion, followed by blood collection through incision of the lateral ventral artery. Each measurement was performed in triplicate to ensure accuracy. The glucose meter and single-use test strips were obtained from Sanbio Biomedical Sensing Co., Ltd. (Changsha, Hunan Province, China). It is critical to note that all fish were fasted for a minimum of 8 h prior to blood glucose measurement, thus adhering to standard protocols for zebrafish studies. Following blood collection, the zebrafish were euthanized through prolonged exposure in the ice bath, ensuring humane treatment in accordance with institutional guidelines.

Zebrafish were euthanized, and blood glucose and CF were measured prior to freezing. Samples were then frozen in liquid nitrogen and stored in a –80 °C freezer prior to subsequent biochemical analysis.

(1) Whole-body Homogenate Preparation for Metabolic and Oxidative Stress Indicators. Whole-body homogenates were prepared for systemic metabolic assessments. Briefly, an appropriate amount of saline was added to the sample at a 1:9 (mL/g) ratio, followed by tissue mincing and cooling with liquid nitrogen. The sample was fully ground using a liquid nitrogen cryo-grinder (Model: JXFSTPRP-24L, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China), centrifuged at 3000 rpm for 10 min, and the supernatant collected for analysis. Glutathione (GSH) and blood lipid samples were prepared using the same method, with kit-specific extraction buffers provided by the respective commercial kits. Several biochemical markers in the whole-body homogenates were evaluated, as described below. Metabolic indicators: total cholesterol (T-CHO, #A111-1-1), low-density lipoprotein cholesterol (LDL-C, #A113-1-1), high-density lipoprotein cholesterol (HDL-C, #A112-1-1) (Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, Jiangsu province, China). Energy and hormonal markers: insulin and ATP (Shenzhen Zike Biotechnology Co., Ltd., #A016-2, #ZK-F6251, Shenzhen, China). Oxidative stress markers: superoxide dismutase (SOD) (Shenzhen Zike Biotechnology Co., Ltd., Shenzhen, China) and GSH (Jiangsu Keygen Biotechnology Co., Ltd., Nanjing, Jiangsu province, China).

(2) Liver Mitochondrial Isolation and Functional Analysis. To evaluate mitochondrial function, liver mitochondria were isolated using a kit to prepare a mitochondrial suspension (#KGA829, Jiangsu Keygen Biotechnology Co., Ltd., Nanjing, Jiangsu province, China). Briefly, liver tissues were digested in an enzyme-containing buffer at 37 °C for 2–4 h with gentle shaking (120 rpm). The suspension was filtered through a 200-mesh sieve (Dongguan City Torch Feng Screen Products Co., Ltd., Dongwan, Guangdong province, China), washed twice, and aliquoted. ROS were labeled with 2^′,7^′-Dichlorodihydrofluorescein diacetate (DCFH-DA, diluted 1000 times) fluorescent probe (S0033S, Shanghai Bio-Tech Biotechnology Co., Ltd., Shanghai, China) and quantified by flow cytometry (FCM). To determine the membrane potential ($\mathrm{\Delta }$$\mathrm{\Psi }$m), mitochondria were stained with 5,5^′,6,6^′-Tetrachloro-1,1^′,3,3^′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, diluted 1000 times) dye (M8650, Solarbio, Beijing, China) and analyzed by FCM after incubation at 37 °C for 20 min.

Zebrafish biopsy specimens (1 mm $\times$ 1 mm $\times$ 1 mm) were fixed in 2.5% glutaraldehyde solution (#PH9003, Fuzhou Feijing Biological Technology Co., Ltd., Fuzhou, Fujian Province, China) and stored overnight at 4 °C. After further fixation with 1% Osmic acid (#TO030810ML, Sinopharm Group Chemical reagent Co., Ltd., Shanghai, China) for 1 h and dehydration with graded ethanol (30% Mel 100%, #10009218, Sinopharm Group Chemical reagent Co., Ltd., Shanghai, China), samples were treated with a mixture of embedded agent and acetone (vol:vol of 1:1, #10000418, acetone was purchased from Sinopharm Group Chemical reagent Co., Ltd., Shanghai, China) for 1 h, followed by a mixture of embedding agent and acetone (vol:vol of 3:1) for 3 h, and finally pure embedding agent overnight. The infiltrated sample was heated overnight at 70 °C for final embedding. Samples were cut with a Leica EMUC7 ultra-thin slicer (Leica Microsystems GmbH, Wetzlar, Germany) to obtain 70~90 nm thin sections. Samples were stained for 5 min with lead citrate solution (#1722582; Zhongjingkeyi Technology Co., Ltd., Beijing, China) and hydrogen peroxide–acetic acid solution, freshly prepared by mixing 3% hydrogen peroxide (#73113762) with 5% glacial acetic acid (#FA3550002) at a 1:1 ratio (both from Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China). After drying, the sections were observed by transmission electron microscopy (TEM, FEI Tecnai G2 F30, FEI Company, Hillsboro, OR, USA) at a voltage of 100 KV [27].

ROS and JC-1 were analyzed using flow cytometry and its software (NovoCyte, version 8.0.2, Agilent, Santa Clara, CA, USA) [28]. For DCFH analysis, the qualitative of ROS levels was based on the fluorescence intensity distribution, where the peak shift in histograms reflects changes in ROS levels. In addition, standardized criteria were established to evaluate the degree of peak shift for each group, enabling a clear distinction between increases and decreases in ROS levels. For quantitative analysis, we used the peak fluorescence intensity values, which allowed for objective comparison of ROS levels across groups. Similarly, for JC-1 analysis, a quadrant gate in NovoCyte was utilized to distinguish between monomeric (green fluorescence) and aggregated (red fluorescence) forms of the dye. Within the standard quadrants, data in the lower-right quadrant reflects the degree of mitochondrial membrane potential depolarization.

Biochemical indicators were analyzed using GraphPad Prism 8.0.2 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as the mean $\pm$ standard deviation. The normality of data distribution was assessed using the Shapiro-Wilk test. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used for normally distributed data. Data that did not adhere to a normal distribution were analyzed using the Mann-Whitney U test, with results displayed as box plots to illustrate the data distribution. For multiple group comparisons, a one-way ANOVA followed by Tukey’s post-hoc test was performed for normally distributed data, while the Kruskal-Wallis test was applied for non-normally distributed data. A significance level of p 0.05 was considered to be statistically significant. Here, # denotes the results of significance comparisons between the control group and the two T2DM groups, while * denotes the results of significance comparisons between the T2DM and T2DM+ST groups. Significance thresholds were marked as follows: p 0.05 (* or #), p 0.01 (** or ##), p 0.001 (*** or ###), and p 0.0001 (**** or ####).

The results presented in Fig. 3A show that ST ameliorates hyperglycemia and insulin resistance in zebrafish. The fasting blood glucose level in the control group was relatively stable, with individual values of between 1.5 mmol/L and 3.5 mmol/L. In contrast, zebrafish in the T2DM group exhibited significantly elevated fasting blood glucose levels, ranging from 3.7 mmol/L to 7.8 mmol/L (p 0.0001). After exercise intervention (T2DM+ST group), the blood glucose level decreased significantly (p 0.001), with individual values of between 2.4 mmol/L and 4.1 mmol/L. A small number of zebrafish showed a return to the normal level. Correspondingly, the T2DM group had a significantly increased level of insulin compared to the control group (p 0.0001). ST induced a down-regulation of insulin levels compared to the T2DM group (p 0.05), suggesting enhanced insulin sensitivity in T2DM+ST zebrafish and optimized utilization of the available insulin to regulate blood glucose (Fig. 3B).

Fig. 3.

T2DM zebrafish groups display different degrees of blood sugar and blood lipid metabolism disorders. (A) Blood sugar. (B) Insulin. (C) Condition factor (CF). (D) Low-density lipoprotein cholesterol (LDL-C). (E) High-density lipoprotein cholesterol (HDL-C). (F) Total cholesterol (T-CHO). # denotes a significant difference between the control group and the other groups, while * denotes a significant difference between the T2DM and T2DM+ST groups. Significance thresholds are marked as follows: ns p $>$ 0.05, p 0.05 (*), p 0.01 (** or ##), p 0.001 (*** or ###), and p 0.0001 (####).

Given the frequent association of T2DM with insulin resistance and lipid metabolic anomalies, we compared the blood lipid profiles across the three groups. The CF level in the T2DM group was significantly higher than in the control group (p 0.0001). However, ST did not reduce the CF level in T2DM zebrafish (Fig. 3C, p $>$ 0.05). The T2DM group displayed significantly increased LDL-C and T-CHO levels (p 0.001 and p 0.01, respectively) compared to the control group, and a non-significant reduction in HDL-C. The T2DM+ST group showed significantly decreased LDL-C and T-CHO levels compared to the T2DM group (p 0.01), and significantly increased HDL-C (Fig. 3D–F, p $>$ 0.05). These findings underscore the potential for ST in enhancing HDL-mediated cholesterol reverse transport and in mitigating LDL levels, thereby averting diabetes progression and cardiovascular complications.

As expected, a significant increase in ROS production was observed in the liver of T2DM zebrafish. Furthermore, ST reduced ROS production in the liver of T2DM zebrafish by an average of 28% (Fig. 4A,B, p 0.05). After modeling, the levels of SOD and GSH in the T2DM group decreased by 59.5% and 46.2%, respectively, compared with the control group (p 0.0001). However, these levels increased significantly after exercise by 48.8% and 4.8%, respectively, compared with the control group (p 0.0001 for SOD; p $>$ 0.05 for GSH), and were restored to levels close to or higher than those of the control group (Fig. 4C,D). These findings highlight the potential for exercise in rectifying oxidative stress imbalances in diabetic models.

Fig. 4.

High glucose induces the generation of oxidative stress. (A) Scatter plot of 2^′,7^′-Dichlorodihydrofluorescein (DCFH)-labeled cell clusters in zebrafish liver. (B) Fluorescence intensity analysis of DCFH. (C) Comparative analysis of superoxide dismutase (SOD) content. (D) Comparative analysis of GSH content. # denotes a significant difference between the control group and the other groups, while * denotes a significant difference between the T2DM and T2DM+ST groups. Significance thresholds are marked as follows: ns p $>$ 0.05, p 0.05 (#), p 0.01 (** or ##), p 0.001 (***), and p 0.0001 (**** or ####).

To further assess the impact of ST on mitochondrial dysfunction, the mitochondrial membrane potential (MMP) and ATP levels were evaluated in zebrafish across the experimental groups. JC-1 staining was used to evaluate MMP. A higher proportion of cells in Q3–4 (green fluorescence) indicates lower mitochondrial membrane potential, which is consistent with mitochondrial depolarization. The T2DM group exhibited a significant decrease in MMP of 68.6% compared to the control group (Fig. 5A,B, p 0.0001). Furthermore, the T2DM group showed increased ATP levels compared to the control group (Fig. 5C, p 0.001). A marked recovery in MMP was observed in the T2DM+ST group (p 0.0001). Additionally, ATP levels were significantly elevated in the T2DM+ST group. These results indicate that ST led to improvements in both MMP and ATP production in zebrafish.

Fig. 5.

Mitochondrial membrane potential and adenosine triphosphate (ATP) levels in zebrafish liver tissue. (A) Scatter plot of cell clusters labeled with 5,5^′,6,6^′-Tetrachloro-1,1^′,3,3^′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) in zebrafish liver. JC-1 staining was used to evaluate the mitochondrial membrane potential (MMP). (B) Fluorescence intensity analysis of JC-1. (C) Comparative analysis of ATP content. # denotes a significant difference between the control group and the other groups, while * denotes a significant difference between the T2DM and T2DM+ST groups. Significance thresholds are marked as follows: p 0.05 (#), p 0.01 (**), p 0.001 (###), and p 0.0001 (**** or ####).

The T2DM group displayed a plethora of mitochondrial anomalies in their liver myofibrils, characterized by heterogeneous shapes and sizes (Fig. 6). These mitochondria predominantly exhibited flattened or elongated cristae, accompanied by the presence of numerous myeloid bodies within. A majority of the mitochondria in randomly selected microscopic fields of T2DM zebrafish liver exhibited disrupted double-membrane structures (white arrow in Fig. 6), indicating the presence of significant structural damage. Concurrently, instances of mitochondrial fusion and fission were observed (black arrow). In comparison, zebrafish in the T2DM+ST group showed greatly improved mitochondrial structure. A substantial accumulation of $\beta$-like glycogen, presenting as layered aggregates, was observed near the liver mitochondria in the T2DM group. In contrast, T2DM+ST zebrafish exhibited a significant reduction in glycogen accumulation, which manifested as a more diffuse pattern.

Fig. 6.

Transmission electron micrographs of liver mitochondria in zebrafish. G represents accumulated glycogen in the liver. The black arrows indicate the morphology of mitochondrial fusion/fission, while the white arrows indicate damage to the mitochondrial double membrane structure. Scale bar: 500 nm.

The shoal test was employed to assess anxiety-like behavior in zebrafish. Fish tracking software was used to measure the distance between individual fish and the shoal, as well as variations in the average swimming speed (Fig. 7A). The T2DM group displayed a significantly longer average distance from the shoal compared to the control group, indicating reduced social interaction in these zebrafish (Fig. 7B, p 0.001). Additionally, the average swimming speed in the T2DM group was faster than in the control group (Fig. 7C, p 0.01). The average shoal distance was significantly decreased in the T2DM+ST group compared to the T2DM group (p 0.001), while the swimming speed returned to a similar velocity to the control group (p 0.001). These results indicate that ST positively influenced the social and anxiety-like behaviors of T2DM zebrafish.

Fig. 7.

Shoal test results. Ten zebrafish in each group participated in the test, with the trajectories represented by different colors. (A) Schematic diagram of the shoaling test (Created using BioRender.com) and zebrafish trajectory. (B) The average distance between zebrafish in the fish shoal test. (C) The average speed of zebrafish in the shoal test. # denotes a significant difference between the control group and the other groups, while * denotes a significant difference between the T2DM and T2DM+ST groups. Significance thresholds are marked as follows: ns p $>$ 0.05, p 0.01 (##), p 0.001 (*** or ###).

We next created two identical virtual partitions (Fig. 8A) to analyze the exploration patterns (i.e., spatial and temporal dynamics of behavior). Significant differences in the preference for swim zones (up-down distance/time) were observed between the control and T2DM groups. Heatmap results showed that T2DM zebrafish showed a strong preference for the lower region (Fig. 8B–E), whereas zebrafish in the control and T2DM+ST groups generally preferred the upper region (Fig. 8A, p $>$ 0.05 for T2DM+ST groups). Fish in the control, T2DM and T2DM+ST groups showed an average exploration preference for the upper region of 38.05%, 17.60% and 42.17% (distance traveled/total distance traveled in the upper region), respectively (Fig. 8F). Moreover, the T2DM+ST group tended to swim near the surface of the water, spending an average of 39.34% of the total observation time in the upper area (calculated as the time spent in the upper area divided by the total observation time), compared to 32.63% in the control group and 14.55% in the T2DM groups. The average transfer frequency in the T2DM group (9.81 times) was markedly reduced compared to the control group (17.22 times, p 0.05), and significantly lower than that of the T2DM+ST group (27.59 times, p 0.01). Moreover, the delay in entering the upper subregion for the first time was surprisingly long in the T2DM group, with no zebrafish exploring this area in less than 10 sec. We next normalized the preference of each zebrafish group for the upper layer as follows: swimming distance in the upper layer/total driving distance. The results showed that T2DM zebrafish had a markedly reduced preference for the upper layer compared to the lower layer (Fig. 8F–H). Although some differences for this parameter were observed with the control group, none were found for the T2DM+ST group.

Fig. 8.

Novel tank test (NTT) results. Ten zebrafish in each group participated in the test. (A) The movement trajectory of zebrafish and a diagram of the test device (Created using BioRender.com). (B) Swimming time in the top region. (C) Driving distance in the top region. (D) Latency (time required for first access to the top). (E) The number of shuttles between the top and bottom region. (F) Preference of zebrafish for top region. (G) Comparison of the distance traveled by each zebrafish in the top and bottom regions. (H) Comparison of time spent in the top and bottom regions for each zebrafish. # denotes a significant difference between the control group and the other groups, while * denotes a significant difference between the T2DM and T2DM+ST groups. Significance thresholds are marked as follows: ns p $>$ 0.05, p 0.05 (* or #), p 0.01 (** or ##), p 0.001 (*** or ###), and p 0.0001 (****).