*3.3. Diazoxide and Exercise Decrease ROS Levels and Lipid Peroxidation in Slow and Fast Skeletal Muscle of Obese Rats*

To analyze the effect of diazoxide treatment and the combination of both on the oxidative stress of muscle tissue, ROS levels, and lipid peroxidation of the different groups' soleus and EDL muscles were evaluated. Figure 2 shows ROS and lipid peroxidation levels of the soleus muscle (Figure 2A,C) and EDL (Figure 2B,D) before and after fatigue. In the evaluation of ROS in the soleus muscle (Figure 2A) and the EDL muscle (Figure 2B), there was an increase in ROS levels of 51.96% and 35.89% before fatigue and 40.81% and 81.41% after fatigue, respectively, in the group of obese rats compared to the control group. However, a decrease in ROS levels with each of the treatments was observed; in the obese group treated with diazoxide, there was a decrease in ROS levels of 34.80% for soleus muscle and 45.46% for EDL muscle before fatigue, and 26.67% for soleus muscle and 29.26% after fatigue. The exercised obese group showed a decrease of 38.69% in soleus muscle and 28.59% for EDL muscle before fatigue and 58.57% in soleus muscle and 71.73% for EDL muscle after fatigue and finally in the

group of obese rats exercised with diazoxide, a decrease of 39.95% for soleus muscle and 37.25% for EDL muscle before fatigue and 63.49% for soleus muscle and 69.38% for EDL muscle after fatigue, all compared to the group of obese rats without any treatment.

In lipid peroxidation, the results of the soleus muscle (Figure 2C) and EDL muscle (Figure 2D) showed an increase in lipid peroxidation levels of 70.91% before fatigue and 70.19% after fatigue and 43.48% before fatigue and 51.19% and after fatigue, respectively, in the group of obese rats compared to the control group. However, a decrease in lipid peroxidation levels was observed with each of the treatments separately and a synergistic effect with both. In the group of obese rats treated with diazoxide, a decrease of 26.91% was observed for soleus muscle and 19.70% for EDL muscle before fatigue, and 17.24% for soleus muscle and 23.91% for EDL muscle after fatigue. The group of exercised obese rats showed a decrease of 22.84% for the soleus muscle and 21.07% for the EDL muscle before fatigue, and 38.36% for the soleus muscle and 35.87% for the EDL muscle. Finally, in the group of obese rats exercised with diazoxide, a decrease of 39.96% was observed for soleus muscle and of 31.46% for EDL muscle before fatigue and 44.64% for soleus muscle and 43.77% for EDL muscle after fatigue, concerning the obese group without treatment.

**Figure 2.** Effect of diazoxide and exercise on ROS levels and lipid peroxidation in slow and fast skeletal muscle of obese rats. (**A**) ROS levels in the soleus muscle, (**B**) ROS levels in the EDL muscle, (**C**) TBARS levels in the soleus muscle, and (**D**) TBARS levels in the EDL 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.
