Mechanism for exercise-mediated prevention against muscle wasting on extensor digitorum longus muscle in Spontaneously Diabetic Torii fatty rats

We previously reported the significant increase in limb muscle strength and cross-sectional area of the type IIb muscle fibers in the extensor digitorum longus (EDL) muscle in a type 2 diabetic animal model, with Spontaneously Diabetic Torii (SDT) fatty rats (n = 6) undergoing regular treadmill exercise from 8 to 16 weeks of age compared with sedentary SDT fatty rats (n = 6). This study investigated the mechanism by which exercise training prevented skeletal muscle wasting in the EDL muscle of the SDT fatty rats. The endurance exercise for 8 weeks downregulated the expression of muscle RING-finger protein-1 (an E3 ubiquitin ligase) and upregulated the expression of CD31, insulin receptor substrate-2, and phosphorylated endothelial nitric oxide synthase in the EDL muscle of 16-week-old SDT fatty rats. Endurance exercise training might reduce muscle wasting by preventing muscle degradation and increasing the angiogenic response in the EDL muscle in type 2 diabetes. Supplementary Information The online version contains supplementary material available at 10.1186/s12576-023-00865-5.


Muscle strength during an observational period
Muscle strength was measured every 4 weeks from ages 8 to 16 weeks using the forelimb grip test with a grip strength meter (MK-380CM/FM; Muromachi Kikai, Co., Ltd., Tokyo, Japan).
The average of three measurements of muscle strength per animal per time point was used for the comparative analysis.The muscle strengths of all the rats were normalized to their body weight.The mean muscle strength was weaker in the SDT fatty group than in the SD group, but not significantly.Although no significant changes were found throughout the experimental period in each group, the muscle strength was significantly weaker in the SDT-Cont group than in the SD group at ages 12 and 16 weeks.In the SDT-Ex group, muscle strength was significantly greater than in the SDT-Cont group at 12-and 16-week-old, although it was significantly lower than in SD-Ex group at age 16 weeks (Table S1).These method and results were reported in detail in reference No.18.

Muscle weight in 16-week-old SD and SDT fatty rats
The soleus muscle and the extensor digitorum longus (EDL) muscle were removed after 16 hours of starvation at 16-week-old and their weight was measured.The muscle weights of all the rats were normalized to their body weight.Although the muscle weights of the soleus and EDL muscles were significantly lighter in the SDT fatty group than in the SD group, the muscle weight of the EDL muscle was significantly heavier in the SDT-Ex group than in the SDT-Cont group.

Body weight and blood glucose levels during an observational period
Body weight and blood glucose levels were measured in all the rats every 4 weeks from ages 8 to 16 weeks.Body weight of all the SDT fatty rats was significantly heavier than that of all the SD rats at 8-week-old (Fig. S1a).While the body weights of SD-Ex group were significantly lighter than those of the SD-Cont group at 12-and 16-week-old, there were no significant differences between the SDT-Cont and SDT-Ex groups (Fig. S1a).Blood glucose levels were significantly higher in the SDT fatty group compared with the SD group and were similar between the SDT-Cont and SDT-Ex groups (Fig. S1b).These results were reported in reference No.18.

Figure S4
Figure S4 Western blot analysis of p-Foxo1 in EDL (a) and soleus (b) muscles, Foxo1 in EDL (c) and soleus (d) muscles and α-tubulin in EDL (e) and soleus (f) muscles on the same membrane in each group.Red lines represent the edge of each cut membrane.Red boxes show the regions of the original blots used in main figures.

Figure S5
Figure S5 Western blot analysis of p-NF-B in EDL (a) and soleus (b) muscles, NF-B in EDL (c) and soleus (d) muscles and α-tubulin in EDL (e) and soleus (f) muscles on the same membrane in each group.Red lines represent the edge of each cut membrane.Red boxes show the regions of the original blots used in main figures.

Figure S6 Figure S7 Figure S8 Figure S9
Figure S6 Western blot analysis of MuRF1 in EDL (a) and soleus (b) muscles, and α-tubulin in EDL (c) and soleus (d) muscles on the same membrane in each group.Red lines represent the edge of each cut membrane.Red boxes show the regions of the original blots used in main figures.

Figure S10
Figure S10 Western blot analysis of p-eNOS in EDL (a) and soleus (b) muscles, eNOS in EDL (c) and soleus (d) muscles, and α-tubulin in EDL (e) and soleus (f) muscles on the same membrane in each group.Red lines represent the edge of each cut membrane.Red boxes show the regions of the original blots used in main figures.

Table S1 Muscle strength at 8-, 12-, and 16-week-old.
All data were expressed as the mean (SEM).** P < 0.01 versus the SD-Cont group at the same age; ## P < 0.01 versus the SD-Ex group at the same age; § P < 0.05 and § § P < 0.01 versus the SDT-Cont group at the same age.This table was created based on the results of reference No.18.

Table S2 Muscle weight in 16-week-old SD and SDT fatty rats Muscle weight per body weight, g/kg SD-Cont group,
**, ## 0.61 (0.01) **, ##, § All data were expressed as the mean (SEM).** P < 0.01 versus the SD-Cont group at the same age; ## P < 0.01 versus the SD-Ex group at the same age; § P < 0.05 versus the SDT-Cont group at the same age.This table was created based on the results of reference No.18.