*3.4. E*ff*ect of Diazoxide and Exercise on Glutathione Redox Status in Slow and Fast Skeletal Muscle of Obese Rats*

To evaluate the effect of diazoxide, exercise, and both on glutathione's redox status in obesity, Figures 3 and 4 show the results obtained for total glutathione levels (GSH + GSSG), reduced glutathione (GSH), and oxidized glutathione (GSSG) before and after inducing fatigue for the soleus muscle and EDL muscle, respectively. These results indicate that during obesity no differences were observed in the levels of GSH + GSSG before and after fatigue in relation to the control group in the soleus muscle (Figure 3A), while in the EDL muscle (Figure 4A) a decrease in GSH + GSSG levels of 16.74% before fatigue and 20.12% after fatigue was observed in relation to the control group. However, in the group of obese rats treated with diazoxide, a 45.31% increase was observed for soleus muscle and 49.38% for EDL muscle before fatigue and 88.90% for soleus muscle and 37.48% for EDL muscle after fatigue. The group of obese rats exercised showed an increase of 18.80% before fatigue for soleus muscle and 24.37% for EDL muscle and 71.42% for soleus muscle and 51.38% for EDL after fatigue. Finally, in the group of obese rats exercised with diazoxide, an increase of 28.16% was observed for soleus muscle and 27.27% for EDL muscle before fatigue and 92.23% for soleus muscle and 83.33% for EDL muscle after fatigue in relation to the group of obese rats without treatment. Regarding the GSH levels of the soleus muscle (Figure 3B) and EDL (Figure 4B), the group of obese rats showed a decrease in levels of 39.76% for soleus muscle and 57.84% for EDL muscle before fatigue and a decrease of 58.25% for soleus muscle and 52.20% for EDL muscle after fatigue to control. However, an increase in GSH levels could be seen in obese rats with each of the treatments, concerning obese rats without treatment.

**Figure 3.** Effect of diazoxide and exercise on glutathione redox status in slow skeletal muscle of obese rats. (**A**) Total glutathione soleus muscle, (**B**) reduced soleus glutathione muscle, and (**C**) oxidized glutathione soleus muscle, before and after fatigue. C: control; D: diazoxide; E: exercise; O: obese; OD: obese diazoxide; OE: obese exercise; OED, obese exercise with diazoxide; ED: exercise with diazoxide. Data are represented as the mean ± standard error. Different letters indicate statistically significant differences between the groups, capital letters compare the different groups before fatigue, and lowercase letters compare the different groups after fatigue. \* indicates significant differences in the comparison of the same group before and after fatigue (*p* < 0.05) two-way ANOVA, Tukey's post-hoc test, *n* = 8.

**Figure 4.** Effect of diazoxide and exercise on glutathione redox status in fast skeletal muscle of obese rats. (**A**) EDL muscle with total glutathione, (**B**) EDL muscle with reduced glutathione, and (**C**) EDL muscle with oxidized glutathione, before and after fatigue. C: control; D: diazoxide; E: exercise; O: obese; OD: obese diazoxide; OE: obese exercise; OED, obese exercise with diazoxide; ED: exercise with diazoxide. Data are represented as the mean ± standard error. Different letters indicate statistically significant differences between the groups, capital letters compare the different groups before fatigue, and lowercase letters compare the different groups after fatigue. \* indicates significant differences in the comparison of the same group before and after fatigue (*p* < 0.05) two-way ANOVA, Tukey's post-hoc test, *n* = 8.

To conclude, it should be mentioned that the levels of oxidized glutathione did not show significant differences between the muscles analyzed before and after fatigue in both muscles of each group evaluated.

#### **4. Discussion**

More than a third of the world's adult population suffers from obesity [30], with more than 500 million people worldwide. Obesity is related to many pathologies [7] and is exacerbated by a sedentary lifestyle and lack of physical activity (WHO, 2016) [31]. Skeletal muscle dysfunction is a complication of obesity, as it causes significant atrophy, leading to a decrease in the muscle contraction force, reduced ability to support fatigue, and numerous metabolic alterations and increased oxidative stress [1,3,6].

Studies conducted by Alemzadeh et al. [17], Pompeani et al. [1], Bae et al. [5], and Lu et al. [32] showed that obesity-induced with a high-fat diet increases bodyweight and plasma glycemia in Wistar rats. The results in Table 3 show consistency with these studies for both parameters. However, in this study, it was observed that both the treatment with diazoxide, the exercise protocol, and the combination of both decreased bodyweight and plasma glycemia during obesity, effects that may be related to the reduction in the consumption of food, improvement of basal metabolic rate, reduction of lipogenesis, improvement of insulin sensitivity and glucose transport, and suppression of hyperinsulinemia [5,17,22,33].

A higher concentration of lipids in muscle tissue produces skeletal muscle dysfunction [6]. This experimental series observed how muscle contraction and fatigue resistance were decreased in the soleus muscle and EDL of obese rats (Figure 1). The results showed that obesity affects the normal contractile function of muscles, which is associated with an increase in intramuscular fat,

causing a deficiency in the muscle ability to contract [6], an increase in proteolysis, change in fiber type, metabolic alterations, and increased oxidative stress [1,7,34].

Previous studies have shown that treatment with diazoxide prevents and reverses metabolic disorders, such as loss of insulin sensitivity, and has been associated with an improvement in glucose transport and the promotion of lipid metabolism [6], which directly or indirectly improves the functioning of muscle tissue.

In this study, it was observed that the treatment with diazoxide increased muscle contraction and promoted resistance to fatigue in the obese group treated with this drug (Figure 1); data are consistent with what was observed by García et al. [18] where diazoxide increased the post-fatigue tension of the mouse EDL muscle. This suggests that the protective effect of diazoxide in obesity may be due to the opening of KATP channels, leading to an increase in cellular respiration through the electron transport chain (CTE) and an increase in the synthesis of ATP [35], favoring muscle contraction and resistance to fatigue [36,37]. A very similar effect was observed with implementing the exercise protocol during obesity (Figure 1). Studies by Zigman et al. [38] and Kraljievic et al. [13] showed an exercise-induced increase in the expression of cardiac sarcolemmal KATP channels, which improved conditioning and contraction, a process that is not ruled out could be occurring in our results, as improvement in muscle contraction and fatigue resistance. Similarly, the positive effects of exercise are attributed to the decrease in intramuscular fat, as a consequence of the increase in skeletal muscle metabolism [3], or the regulation of numerous pathways of signaling in which exercise participates by improving its functioning, such as insulin sensitivity, glucose transport, lipid profile, and reduced stress markers [3,5].

The increase in fat stored in skeletal muscle has been associated with increased ROS levels, the appearance of oxidative stress, and eventual cell damage [7].

Abrigo et al. [2] evaluated ROS levels in obese mouse muscles induced by a high-fat diet. They observed in this group an increase in ROS levels 7.22 times that of the control group. This is consistent with the obtained results in this work since it was possible to observe increased ROS levels in the groups of obese rats, compared to the control group in both muscles before and after the fatigue was induced (Figure 2A,B), observing a higher level of ROS in EDL muscle because fast-twitch fibers are more susceptible to oxidative stress than slow-twitch fibers [39]. The increase in ROS levels during obesity is due to the increase in intramuscular fat causing a reduction in the muscle's ability to contract [6], which causes reduced ATP levels and decreases mitochondrial volume producing mitochondrial dysfunction and promoting muscle fatigue [36,37,39]. It has been shown that there is a dose-dependent relationship between ROS concentration and reduced muscle contractility and the occurrence of fatigue [40]. This work showed that the use of diazoxide, regular exercise, and the combination of both reduced ROS levels in the group of obese rats in both soleus muscle and EDL muscle was analyzed before and after inducing fatigue (Figure 2). A similar effect could be observed in the work of García et al. [18] in mouse muscle fibers where in the presence of diazoxide the ROS production rate was lower compared to the untreated group. This effect was attributed to the opening of mitoKATP channels, which improves contraction and decreases fatigue, thus reducing ROS levels [18,41].

On the other hand, diazoxide can also decrease ROS levels in obesity by preventing mitochondrial dysfunction produced by hyperinsulinemia and hyperglycemia triggered by increased intramuscular fat [5–7]. This protective effect results from inhibiting insulin secretion and improving insulin sensitivity and glucose transport [18], which is also attributed to the characteristic of increasing antioxidant defenses, which favors the decrease of ROS levels [42,43]. In the same way, it was observed how exercise decreased ROS levels in groups of obese rats (Figure 2A,B). This effect of exercise was also observed in the study by Ji et al. [23], where a group of rats was subjected to an exercise regimen where decreased ROS levels were observed compared to the untrained group, in response to an adaptation in their antioxidant systems. This protective role of exercise in obesity could be attributed to the production and activity of antioxidant enzymes, in addition to the activation of numerous signaling cascades involved in exercise [7,23,24].

Similarly, exercise could decrease ROS levels during obesity by increasing the expression and participation of sarcolemmal KATP channels, improving muscle contraction and resistance to fatigue [13,34,38]. In the same way, these positive effects of exercise are attributed to the decrease in intramuscular fat due to the increase in skeletal muscle metabolism [5], or the optimization of signaling cascades related to insulin sensitivity, glucose transport, and inflammatory processes, which in turn decreases ROS production [7,44]. In this experiment series, the muscles of the groups subjected to the exercise protocol analyzed after fatigue showed the same ROS levels as the muscles analyzed before fatigue, an effect that was not observed in the rest of the groups, which can be explained with the concept of hormesis, which describes the response related to a beneficial stress factor at moderate levels and harmful at high levels [4].

It has been observed that one of the main effects of the increase in ROS is the damage to biomolecules [45], so in this study, the levels of TBARS were quantified as an indicator of lipid peroxidation and oxidative stress. This work observed that lipid peroxidation levels were increased in obese rats, both in the soleus and in EDL muscles analyzed before and after inducing fatigue (Figure 2C,D). However, the group of obese rats treated with diazoxide showed decreased lipid peroxidation levels in both muscles (Figure 2C,D). Farahini et al. [46] observed that pretreatment with diazoxide contributed to muscle tissue resistance against ischemic damage by decreasing lipid peroxidation. The same was appreciated by Moghtadaei et al. [42], where they observed this protective effect of diazoxide in skeletal muscle ischemia, as the levels of lipid peroxidation were decreased in the group preconditioned with diazoxide.

Similarly, the experimental series results showed decreased lipid peroxidation levels in the group of obese-exercise rats (Figure 2C,D). This effect with the implementation of an exercise protocol was previously observed by Lambertucci et al. [24], where the levels of lipid peroxidation of muscle tissue from trained rats were decreased. The decrease in both ROS levels and lipid peroxidation with diazoxide treatment and exercise, and the combination of both is directly proportional to muscle contraction improvement and increased resistance time to fatigue.

Cells have antioxidant systems that protect them against oxidative damage. This includes the antioxidant enzyme glutathione peroxidase, the synthesis of which can be modified by exercise, diet, and age [40]. Our results showed that both the levels of GSH + GSSG of the soleus muscle (Figure 3A) and EDL muscle (Figure 4A), as well as of GSH of the soleus muscle (Figure 3B) and EDL muscle (Figure 4B) were decreased during obesity, while the GSSG levels of the soleus muscle (Figure 3C) and EDL muscle (Figure 4C) were increased. The decrease in the content of GSH + GSSG suggests that obesity affects the synthesis of this antioxidant, possibly because ROS that are overproduced during this pathology are involved in activating various signaling pathways that negatively affect the expression of genes involved. The synthesis and content of GSH decreased [47], suggesting that obesity affects the antioxidant capacity. Diazoxide treatment, exercise, and a combination of both increased GSH + GSSG and GSH levels and, to a lesser extent, reduced GSSG levels in obesity. Moghtadaei et al. [42] reported that glutathione activity was increased in skeletal muscle treated with this drug during ischemia-reperfusion, concerning the muscle without treatment.

On the other hand, exercise presented this same protective effect during obesity since it enhances antioxidant activity [24]. Both the protective effect of diazoxide and exercise may be because these treatments regulate signaling pathways for the activation of genes that regulate the synthesis of antioxidant enzymes [42], in addition to the fact that glutathione synthesis is widely dependent on ATP concentrations. Both diazoxide treatment and exercise favor its production [36].

#### **5. Conclusions**

This is the first study, which shows how diazoxide and exercise and the combination of both improve the contraction and functioning of muscle fibers in obesity by reducing oxidative stress. In conclusion, diazoxide and exercise improves muscle contraction in obesity by decreasing ROS levels, lipid peroxidation, and improving the redox status of glutathione.

**Author Contributions:** Conceptualization, M.G.-B., E.S.-D., and R.M.-P.; methodology, M.G.-B. and K.M.M.-C.; formal analysis, M.G.-B., C.C.-R., and R.M.-P.; investigation, M.G.-B., A.S.-M., and A.R.R.-O; resources, R.M.-P; writing—original draft preparation, M.G.-B., C.C.-R., and R.M.-P.; writing—review and editing, M.G.-B., C.C.-R., E.S.-D., and R.M.-P.; supervision, R.M.-P.; project administration, R.M.-P.; funding acquisition, R.M.-P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Coordinación de la Investigación Científica—Universidad Michoacana de San Nicolás de Hidalgo, R.M.P. CIC-UMSNH2019".

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
