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Swimming Exercise Protects against Insulin Resistance via Regulating Oxidative Stress through Nox4 and AKT Signaling in High-Fat Diet-Fed Mice.

1. Introduction

Lifestyle and dietary changes have led to a rapid increase in the prevalence of metabolic syndrome (e.g., obesity, hypertension, hyperlipidemia, and diabetes) in China. Accumulating evidence points to insulin resistance (IR) as a common pathogenic factor of metabolic syndrome, which is considered to be the most critical trigger of all related metabolic disorders. Exercise has attracted increased attention as an effective means of prevention and nondrug intervention for IR [1]; however, the specific mechanism has not been fully clarified.

The human skeletal muscles are responsible for more than 30% of energy consumption in the body, serving as the main peripheral tissue for glycolipid metabolism stimulated by insulin [2] and the main site for the occurrence of IR. Therefore, the skeletal muscle plays a central role in the maintenance of glucose metabolism. The serine/threonine protein kinase (AKT, also known as protein kinase B) pathway plays an important role in insulin signaling transduction, and skeletal muscle IR is frequently associated with inhibition of this pathway. Exercise, as a physiological stimulus, can cause adaptive changes in signal pathways such as AMP-dependent protein kinase (AMPK), mammalian target of rapamycin (mTOR), and AKT pathways, among which, the reactive oxygen species (ROS) and AMPK pathways generated by redox reactions are highly associated with IR. NADPH oxidase 4 (Nox4) is a constituent of the sarcoplasmic reticulum and is most highly expressed in the rat hindlimb skeletal muscle [3]. Several studies have indicated that increased Nox4 levels are associated with higher production of [H.sub.2][O.sub.2] and an increased risk of IR [4, 5]. Recent studies have also shown that activation of AMPK signaling can downregulate the expression of Nox4 [3, 6] and that the ROS generated by the skeletal muscle tissue mainly originates from Nox4 [7, 8]. Moreover, inhibitors of Nox4 can increase insulin sensitivity [9], and exercise can alter Nox4 expression [10-12].

This background suggests that exercise can influence the AMPK pathway to reduce Nox4-induced ROS, and thereby protect against IR. To test this hypothesis, the aim of this study was to elucidate the role of Nox4 in the development of IR and explore the potential signal transduction pathways that may be mediated by changing the Nox4 expression. Toward this end, we generated a high-fat diet-induced IR model in wild-type mice and skeletal muscle-specific human Nox4-transgenic mice ([m-hNox4.sup.Tg) and investigated (1) the effect of the overexpression of Nox4 on IR and (2) the effector signaling pathways regulated by Nox4.

2. Materials and Methods

2.1. Experimental Animals and Grouping. Forty 4-week-old male C57BL/6 mice were purchased from the Shanghai SLAC Laboratory Animal (SLAC, Shanghai, China). Skeletal muscle-specific human Nox4-transgenic mice ([m-hNox4.sup.Tg]) and control mice (CTL) were obtained from Shanghai Model Organisms Center, Inc. (Shanghai, China). After one week of adaptive feeding with normal feed, the C57BL/6 mice were randomly divided into the normal control group (CON group), normal diet plus exercise group (Ex-CON group), high-fat diet model group (HFD group), and high-fat diet plus exercise group (Ex-HFD), with 10 animals in each group and five animals in each cage. The [m-hNox4.sup.Tg] and CTL mice were also randomly divided into two groups each, resulting in the following four groups: [m-hNox4.sup.Tg] group, [m-hNox4.sup.Tg] plus exercise group (EX-[m-hNox4.sup.Tg]), CTL group, and CTL plus exercise group (EX-CTL), with eight mice in each group and four mice in each cage.

After grouping, the CON and Ex-CON groups were fed a normal diet, while the other groups were all fed an HFD (14.1% protein, 25.9% carbohydrate, 60% fat, 5000 Kcal/1000 g, TROPHIC Animal Feed High-Tech Co. Ltd., China) for 8 weeks. The ambient temperature was 24[degrees]C, relative humidity was 45-55%, and a 12 h light/12 h dark cycle was applied each day. Table 1 lists the ingredient of high-fat feed.

2.2. Exercise Schemes. The mice participating in the experimental exercise intervention received adaptive swimming training in a swimming pool (90 cm x 60 cm x 80 cm) for one week in advance and then the swimming training was conducted for 60 min once per day, six times per week for 8 weeks. This training scheme was formulated with reference to a list of correspondence between animal running speed and maximum oxygen uptake developed by Fernando et al. [13].

2.3. Blood Samples Collection and Blood Biochemistry. Twenty hours after the final session of swimming exercise, all mice were anesthetized by single-dose intraperitoneal injection of 10% chloral hydrate (30mg/kg). Blood samples were collected from the abdominal aorta and the serum was separated by centrifugation at 1500 xg for 10 min. The serum glucose level was measured using an autoanalyzer (RT-1904C; Rayto, China). The insulin concentration in the serum was determined using an enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Bioengineering Institute, China).

The homeostatic model assessment of insulin resistance (HOMA-IR) index and insulin sensitivity index (ISI) were calculated according to the following formulas [14, 15]:

HOMA - IR = (FINS x FBG)/22.5, ISI = ln [1/(FINS x FBG)], (1)

where FINS is the fasting insulin level and FBG is the fasting blood glucose level.

2.4. Intraperitoneal Glucose Tolerance Test (IGTT) and Intraperitoneal Insulin Tolerance Test (IITT). To assess whether the mice exhibited alterations in peripheral glucose regulation, IGTTs were performed. The mice were fasted for 12 h prior to baseline glucose readings (taken in duplicate) with subsequent intraperitoneal glucose injection (20% D-glucose at 2 g/kg body weight). Blood was collected from the tail vein and blood glucose readings were taken in duplicate at baseline (time 0), and at 15, 30, 60, 90, and 120 min post glucose injection using a glucometer (ACCU-CHEK Compact Plus, Roche, Germany).

Following the IGTT, the mice were allowed 1 week of recovery prior to the IITT. For insulin tolerance testing, the mice were fasted for 6 h prior to testing and then were delivered an intraperitoneal injection of insulin (2 U/kg body weight, insulin; Sigma, St. Louis, MO, USA) with blood glucose levels monitored, in duplicate, at time point 0 (preinjection), and at post-injection time points of 15, 30, 60, 90, and 120 min.

2.5. Skeletal Muscle Sampling. After intraperitoneal anesthesia with 10% chloral hydrate at a dose of 30 mg/kg body weight, bilateral gastrocnemius muscles were taken, rapidly placed in liquid nitrogen, and stored at -80[degrees]C. Fifty milligrams of the skeletal muscle tissue was mixed with 500 mL of 0.9% normal saline (mass fraction: 10%), and a homogenate of the skeletal muscle tissue was prepared with an ultrasonic processor (40 amperes, 5 s each time at intervals of 10 s, repeated five times). The homogenate was centrifuged at 3000 g at 4[degrees]C for 15 min, and the supernatant was collected and stored at -20[degrees]C for subsequent testing.

2.6. Measurement of Oxidative Stress Parameters in the Skeletal Muscle. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), in the skeletal muscle, were determined with superoxide dismutase determination kit (Sigma) and glutathione peroxidase determination kit (Sigma), respectively, following the manufacturer's instructions. The 8-hydroxyl-deoxyguanosine (8OHdG) content was measured using enzyme-linked immunosorbent assay (ELISA) (R&D, Minneapolis, USA).

2.7. Real-Time Fluorescence Quantitative Polymerase Chain Reaction (RT-qPCR) for AMPK and Nox4 in the Skeletal Muscles. RT-qPCR was used to detect the mRNA levels of AMPK[alpha]2 and Nox4 in the skeletal muscles of mice. TRIzol reagent (Takara) was used to extract total RNA from the skeletal muscle tissue and was reverse-transcribed to cDNA using the ABI 9700 PCR system according to the manufacturer instructions. RT-qPCR was carried out with a Takara reaction mixture and ABI 7500 Real-Time PCR System using GAPDH as a control for normalization. The primer sequences were as follows:


The mRNA levels of AMPK[alpha]2 and Nox4 were determined by the real-time PCR TB Green method in a 20 [micro]L system at 95[degrees]C for 30 s; 40 cycles of 95[degrees]C for 5 s, 60[degrees]C for 34 s, 95[degrees]C for 15 s, 60[degrees]C for 1 min, and 95[degrees]C for 15 s. The [2.sup.-[DELTA][DELTA]Ct] method was used to calculate the relative mRNA expression levels with reference to the GAPDH level.

2.8. Western Blotting for AMPK[alpha]2, Nox4, pAK[T.sup.Ser473], and AKT Proteins in the Skeletal Muscles. Western blot was used to detect the protein expression levels of AMPK[alpha]2, Nox4, phosphorylated AKT at serine 473 (pAK[T.sup.Ser473]), and AKT in the skeletal muscles of mice. The buffer was mixed with the skeletal muscle protein sample at a 1 : 3 ratio and boiled for 5 min at 95[degrees]C. The same volume of a 40 [micro]g protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a vertical electrophoresis apparatus (BioRad, USA) and transferred to a polyvinylidene fluoride membrane (Fermentas, Canada). The membrane was incubated with the primary antibody at 4[degrees]C overnight, washed with phosphate-buffered saline (PBS) three times, and then incubated with the secondary antibody at room temperature for 2 h. The antibodies used in the current study were rabbit monoclonal anti-AMPK[alpha]2 (1: 1000, Cell Signaling Technology, MA, USA), rabbit monoclonal anti-Nox4 (1: 1000, CST), rabbit polyclonal anti-pIRS-1 (Ser473) (1: 1000, CST), rabbit polyclonal anti-AKT (1: 1000, CST), and HRP-linked antirabbit IgG (1: 2000, CST). After washing with PBS, the membrane was taken out and placed in a gel imaging system (Dolphin, USA) for exposure with enhanced chemiluminescence reaction solution (Thermo, USA). AlphaView SA image analysis software was used to analyze and compare the gray levels of the bands.

2.9. Statistical Analysis. The experimental results are expressed as mean [+ or -] standard deviation. SPSS 23.0 was used for statistical analysis. One-way analysis of variance and Student's t-test were performed for intergroup comparison of the data. The least-squares difference method was used for multiple comparisons. P < 0.05 was set as the level of judging a significant difference.

3. Results

3.1. IGTT, IITT, and IR Indices. To evaluate glucose tolerance and insulin sensitivity, the IGTT and IITT were first performed. As shown in Figures 1(a) and 1(b), the IGTT and IITT results demonstrated that the EX-HFD mice had an increase in glucose and insulin tolerance when compared with those of the HFD mice, and the EX-[m-hNox4.sup.Tg] mice exhibited an increase in the glucose and insulin tolerance when compared with those of[m-hNox4.sup.Tg]mice. Furthermore, to assess IR, we detected the FBG and FINS levels to calculate the HOMA-IR and ISI. As shown in Figures 1(c) - 1(f), FBG did not differ between the CON and HFD groups, whereas FINS and HOMA-IR significantly increased (P < 0.01) and ISI significantly decreased (P < 0.01) in the HFD group. In addition, compared with the HFD group, FBG significantly decreased (P < 0.05), FINS and HOMA-IR significantly decreased (P < 0.01), and ISI significantly increased (P < 0.05) in the Ex-HFD group. Compared with the CTL group, there was no change in FBG, whereas FINS and HOMA-IR increased (P < 0.01) and ISI significantly decreased (P < 0.05) in the [m-hNox4.sup.Tg] group. However, the [m-hNox4.sup.Tg] group showed a slight but insignificant increase in FBG, a significant increase in FINS and HOMA-IR (P < 0.01), and a decrease in ISI (P < 0.05) in comparison to those of the EX-[m-hNox4.sup.Tg] group. These results suggested that overexpression of Nox4 in the skeletal muscle could induce IR. Moreover, these results confirmed that an animal model established with feeding an HFD in mice is also suitable for investigating the mechanisms of IR, and an 8-week swimming exercise can improve IR in Nox4Tg mice and HFD-fed mice.

3.2. AMPK[alpha]2 And Nox4 Expressions in the Skeletal Muscle. As shown in Figure 2, long-term HFD feeding resulted in a significant decrease in the mRNA and protein expression levels of AMPK[alpha]2 in the skeletal muscles of mice (P < 0.01), and a significant increase in the mRNA and protein expression levels of Nox4 (P < 0.01) when compared with those of the CON group. Compared with those of the HFD group, the mRNA and protein expression levels of AMPK[alpha]2 in the skeletal muscle of Ex-HFD mice were significantly upregulated (both P < 0.01), and the expression levels of Nox4 were significantly downregulated (P < 0.01). Moreover, compared with the CTL group, the mRNA and protein expression levels of AMPK[alpha]2 were significantly decreased (P < 0.01), and those of Nox4 were significantly increased (P < 0.01) in the [m-hNox4.sup.Tg] group; however, AMPK[alpha]2 levels were upregulated and Nox4 levels were downregulated (P < 0.01) in Ex-[m-hNox4.sup.Tg] mice compared to those in the [m-hNox4.sup.Tg] group. These findings demonstrate that exercise can increase the expression of AMPK[alpha]2 and reduce the Nox4 level in an IR model.

3.3. Oxidative Stress in the Skeletal Muscles. To confirm the generation of oxidative stress in the skeletal muscles after long-term HFD feeding in mice, we also detected the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and 8-hydroxy-2-deoxyguanosine (8-OHdG). Compared to the CON group, the HFD group showed a significant decrease in SOD and GSH-Px and a significant increase in MDA and 8-OHdG levels in the skeletal muscles (Figure 3). After 8 weeks of exercise, the SOD and GSH-Px activities in Ex-HFD mice significantly increased compared with those in the HFD group (P < 0.05 and P < 0.01, respectively), whereas the 8-OHdG and MDA contents significantly decreased (P < 0.05 and P < 0.01, respectively). In addition, the levels of SOD and GSH-Px in the [m-hNox4.sup.Tg] group were significantly decreased compared to those in the CTL group, whereas the MDA and 8-OHdG levels increased (P < 0.05 and P < 0.01, respectively). After 8 weeks of swimming exercise, the SOD and GSH-Px activities in the Ex-[m-hNox4.sup.Tg] group significantly increased compared with those in the [m-hNox4.sup.Tg] group (P < 0.05 and P < 0.01, respectively), whereas the 8-OHdG and MDA levels significantly decreased (P < 0.05 and P < 0.01, respectively). These findings indicate that an 8-week swimming exercise can reduce the level of oxidative stress in the skeletal muscles of mice under HFD-induced IR.

3.4. AKT Protein Expression in the Skeletal Muscles. Since the HFD was shown to induce oxidative stress, we further verified the effects of the AKT signaling pathway on this process by measuring the protein expression of pAK[T.sup.Ser473] and AKT in the skeletal muscle by western blotting. As shown in Figure 4, compared with mice in the CON group, the expression level of pAK[T.sup.Ser473] was significantly decreased in the HFD group (P < 0.01), whereas the Ex-HFD group showed significantly upregulated expression of pAK[T.sup.Ser473] (P < 0.01) when compared with that of the HFD group. In addition, compared with that of the CTL group, the expression level of pAK[T.sup.Ser473] protein in the skeletal muscles of mice in the [m-hNox4.sup.Tg] group was significantly decreased (P < 0.01), and exercise reversed this effect, with a significantly upregulated expression of pAK[T.sup.Ser473] in the Ex-[m-hNox4.sup.Tg] group (P < 0.01) when compared with that of the [m-hNox4.sup.Tg] group. These findings suggest that swimming exercise could activate AKT in the skeletal muscles of mice fed an HFD.

4. Discussion

4.1. Effects of Swimming Exercise on IR in HFD-Fed Mice. C57BL/6 mice are frequently used as animal IR models. Although a hyperinsulinemic-euglycemic clamp is currently recognized as the gold standard for evaluating IR, this method also has certain limitations. The oral glucose tolerance test is a glucose load test that is widely used to assess the function of pancreatic [beta]-cells and the body's ability to regulate blood glucose, thus serving as a basis for judging whether the body has a glucose metabolism disorder. Under normal circumstances, blood glucose will temporarily increase after oral administration of glucose in mice, and then gradually return to the fasting level. However, when glucose tolerance is abnormal, the blood glucose level does not follow this rule, resulting in an increased blood glucose level and rhythm disorder. ISI is strongly correlated with the glucose infusion rate measured by the hyperinsulinemic-euglycemic clamp method, as the gold standard for evaluating IR [16]. Therefore, we used a combination of the mouse IGTT and IITT, HOMA-IR, and ISI to confirm the establishment of the IR animal model and the effect of exercise intervention on IR. The results showed that 8 weeks of high-fat diet could successfully establish the mouse IR model, and 8 weeks of swimming exercise could relieve the progression of IR in the mice to some extent. Previous evidence have demonstrated that Nox4 is a key factor in type 2 diabetes [5, 17, 18]; our study also showed similar result. Simultaneously, we found after swimming exercise intervention that the swimming mice showed a declined Nox4 level in the skeletal muscle. It suggested that Nox4 can be considered a crucial role in IR prevention through swimming exercise. And here, we undertook a Nox4/ROS pathway based on AMPK in skeletal muscle to clarify the mechanism of swimming exercise protect against IR via regulating Nox4/ROS/AKT signaling pathway in high-fat diet mice.

4.2. Role of AMPK/Nox4/ROS in Improvement of IR through Exercise. AMPK mainly functions in regulating the metabolism of energy substances and maintaining the dynamic balance of energy and is thus considered to be the energy switch of cells. Recent studies on skeletal muscles showed that AMPK activity increases with the extension of exercise duration and is positively correlated with exercise intensity [19]. AMPK[alpha] activity was reported to double after moderate-intensity bicycle exercise with 68% V[O.sub.2]max for 5 min, and tripled after 30 min [20]. In addition, AMPK can be significantly activated by moderate or higher exercise intensities and long-term exercise [21]. In line with these previous findings, the present study showed that the expression level of AMPK[alpha]2 in the skeletal muscles of HFD-fed mice was significantly reduced, which significantly recovered and enhanced after 8 weeks of swimming exercise, proving the key role of AMPK as an energy switch.

Recent studies [3, 5] have shown that a high-sugar diet can directly inhibit AMPK activity, thus significantly increasing the production of Nox4. As skeletal muscle ROS are directly produced by Nox4, excessive ROS generation can lead to an inflammatory reaction and abnormal insulin signal transduction, thus inducing IR. Cells grown in high glucose conditions with Nox4 knocked out through RNA interference showed a decrease in ROS production [3, 4]. In addition, further study found that AMPK could negatively regulate the expression of Nox4 [22, 23]. Therefore, the AMPK/Nox4/ROS axis appears to play a key role in the development and progression of IR. A long-term HFD can reduce the body's antioxidant capacity and lead to insufficient removal of ROS, leading to a state of oxidative stress that increases permeability of cell membrane, thus causing oxidative damage to skeletal muscle cells. Previous studies [24-27] have shown that SOD, MDA, GSH-Px, and 8-OH-dG can comprehensively reflect the state of oxidative stress in the body. SOD can specifically remove harmful free radicals in the body, resist the damage of oxygen free radicals to cells, and rapidly repair damaged cells. MDA is the final product of lipid peroxidation and is an important parameter reflecting the potential antioxidant capacity of the body by indirectly reflecting the degree of tissue peroxidation damage. GSH-Px is an important enzyme that is widely distributed in the body, which catalyzes the reduction reaction of reduced GSH to hydrogen peroxide and can protect the integrity of cell membrane structure and functions. 8-OH-dG is a product formed after ROS oxidizes and damages nuclear DNA. It is stable in vivo and will not be further metabolized once formed. Thus, the content of 8-OH-dG in the body can reflect the state of oxidative damage of nuclear DNA, emerging as a new sensitive index and biomarker for evaluating oxidative stress. Therefore, SOD, MDA, GSH-Px, and 8-OH-dG were used to evaluate the ROS level in the skeletal muscles of mice in this study. ROS have been traditionally considered toxic products of metabolism, which, if occurring in a great extent, may cause skeletal or other tissue/organ dysfunction. And studies have showed that physical activity may protect against ROS-linked tissue damage by either modulating the signaling pathways of AMPK, MAPKs, AKT, or ROS-sensitive transcription factors such as nuclear transcription factor-activated protein 1 (AP-1), peroxisome proliferator-activated receptor gamma coactivator-1[alpha] (PGC-1[alpha]), and nuclear factor erythroid 2-related factor 2 (Nrf2) [28-31]. Physical exercise is regarded as a beneficial role in regulating ROS; however, "exercise induced oxidative damage" is possible to occur if muscle inadaptation was induced by training [32]. The results demonstrated an adaptive response during the swimming exercise intervention in IR through detecting the level of AMPK, SOD, MDA, GSH-Px, 8-OH-dG, and Nox4 in the skeletal muscles of mice It suggested that long-term swimming exercise can improve IR through AMPK/Nox4/ROS pathway.

The PI3K/AKT pathway is highly associated with IR, and a decrease of AKT activity can easily lead to the occurrence and progression of IR. Exercise (treadmill running, swimming, etc.) can activate PI3K/AKT and enhance the associated signal transduction [33]. In addition, significant reductions in the protein expression levels of PI3K and AKT in the skeletal muscle cells of HFD-fed mice have been reported, suggesting abnormal PI3K/AKT signaling [34]. Consistently, we found a decrease in the phosphorylation level of [AKT.sup.Ser473] in the skeletal muscles of HFD-fed mice, which was recovered and enhanced by 8 weeks of swimming exercise. Therefore, exercise can activate AKT pathways in the skeletal muscles to reduce ROS production via Nox4 and protect against HFD-induced IR in mice. These findings was similar to the previous research [4].

In summary, the results of this study provide new insight into the possible mechanism by which exercise can improve IR. An 8-week swimming exercise downregulated Nox4 expression by enhancing the expression level of AMPK in the skeletal muscles, thus preventing the generation of ROS, further enhancing PI3K/AKT insulin signal transduction, and finally relieving IR.

Data Availability

The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest

The authors declare that they have no conflict of interests.

Authors' Contributions

Jun Zhang and Jie Qi contributed to data analysis and developed and wrote the paper. Bo Yang developed the experimental design, contributed to data generation, and reviewed the paper. Xue Luo and Zhichao Ma contributed to the data analysis and paper writing. Bo Zhang and Shuyan Li conducted the mRNA and protein expression analysis in the skeletal muscle. Xuyang Duan contributed to the animal model establishment. Jie Qi, Xue Luo, Zhichao Ma, and Bo Zhang contributed to the work equally.


We thank all of the research staff and students who assisted with animal sampling and provided technical support. This study was supported financially by the National Natural Science Foundation of China (Grant no. 31701037).


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Jie Qi V Xue Luo, (2) Zhichao Ma, (3) Bo Zhang, (1) Shuyan Li, (4) Xuyang Duan, (1) Bo Yang, (5) and Jun Zhang (1)

(1) College of Physical Education, Shanghai Normal University, Shanghai 200234, China

(2) Medical College of Yangzhou Polytechnic College, Jiangsu 225009, China

(3) The School of Physical Education, Wuhan Business University, Hubei 430056, China

(4) College of Physical Education, Yangzhou University, Jiangsu 225009, China

(5) College of Public Health and Management, Wenzhou Medical University, Zhejiang 325000, China

Correspondence should be addressed to Bo Yang; and Jun Zhang;

Received 21 September 2019; Accepted 26 December 2019; Published 21 January 2020

Guest Editor: Amy L. Hui

Caption: Figure 1: Evaluation of insulin resistance. (a) IGTT curves of mice in each group. (b) IITT curves of mice in various groups. (c) The fasting blood glucose in three groups at the 8th week. There were no significant differences in the fasting blood glucose measured after an 8-week swimming exercise intervention compared with the untreated high-fat diet group. (d) The fasting blood insulin in three groups at the 8th week. There was a significant decrease in the fasting blood insulin that was measured after an 8-week swimming exercise intervention compared with the untreated high-fat diet group. (e, f) HOMA-IR and ISI values of different groups at the 8th week, which were also selected to evaluate IR. After training with swimming exercise, HOMA-IR and ISI were both improved. Data are presented as mean [+ or -] SD. ** P < 0.01 versus the CON group; (##) P < 0.01 versus the HFD group. (@@) P < 0.01 versus the CTL group; ([DELTA][DELTA]) P < 0.01 versus the [m-hNox4.sup.Tg] group.

Caption: Figure 2: Effect of swimming exercise on the expression of AMPK[alpha]2 and Nox4 in the skeletal muscle. (a) The mRNA expression of AMPK[alpha]2 and Nox4. (b, c) Protein expressions of AMPK[alpha]2 and Nox4 levels in the skeletal muscle were tested by western blot. Data are presented as mean [+ or -] SD. ** P < 0.01 versus the CON group; (##) P < 0.01 versus the HFD group. (@@) P < 0.01 versus the CTL group; ([DELTA][DELTA]) P < 0.01 versus the [m-hNox4.sup.Tg] group; ([DELTA]) P < 0.05 versus the [m-hNox4.sup.Tg] group.

Caption: Figure 3: Changes in ROS level in skeletal muscles. (a) Swimming exercise upregulated the content of SOD. (b) Swimming exercise increased the level of GSH-Px. (c) Swimming exercise reduced the content of 8-OH-dG. (d) Swimming exercise decreased the level of MDA. Data are presented as mean [+ or -] SD. ** P < 0.01 versus the CON group; * P < 0.05 versus the CON group; (##) P < 0.01 versus the HFD group. (@@) P < 0.01 versus the CTL group; (@) P < 0.05 versus the CTL group; ([DELTA][DELTA]) P < 0.01 versus the [m-hNox4.sup.Tg] group; ([DELTA]) P < 0.05 versus the [m-hNox4.sup.Tg] group.

Caption: Figure 4: Effect of swimming exercise on the protein expression of pAK[T.sup.Ser473] and AKT in the skeletal muscles of mice. Data are presented as mean [+ or -] SD. ** P < 0.01 versus the CON group; (##) P < 0.01 versus the HFD group. (@@) P < 0.01 versus the CTL group; (@) P < 0.05 versus the CTL group; ([DELTA][DELTA]) P < 0.01 versus the [m-hNox4.sup.Tg] group.
Table 1: The ingredient of the high-fat diet.

Ingredient                    Weight (g/kg)

Casein                             195
Maltodextrin                       225
Sucrose                            89
Soybean oil                        33
Lard                               301
Cellulose                          69
Mineral mix, M1021                 68
Vitamin mix, V1010                 14
L-Cystine                           3
Choline bitartrate                  3
Tertiary butylhydroquinone        0.067
Total                             1000
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Article Details
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Title Annotation:Research Article
Author:Qi, Jie; Luo, Xue; Ma, Zhichao; Zhang, Bo; Li, Shuyan; Duan, Xuyang; Yang, Bo; Zhang, Jun
Publication:Journal of Diabetes Research
Geographic Code:9CHIN
Date:Mar 1, 2020
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