2.5. Ex vivo EDL and soleus muscle contractility assay

Isolated soleus and extensor digitorum longus (EDL) muscles from male mice (12–16 weeks of age) were used for muscle contractility assays. Muscles were incubated at 30 °C in oxygenated (95% O2 and 5% CO2) physiological salt solution as previously described in [27] for Figure 1M–P and in [28] for Figure 1Q–T. The buffer was continuously oxygenated with 95% O2 and 5% CO2 during the incubation period and maintained at 30 °C. For results shown in Figure 1M–P, muscles were set to an initial resting tension of 10 mN (optimal length, L0; 300B, Aurora Scientific) and maintained by a stepper motor. Dynamic Muscle Control software (DMC Version 4.1.6, Aurora Scientific) was used to control the force and position inputs of the servomotor arm and the stepper motor. For results shown in Figure 1M–O, the stimulated muscle was subjected to 3 isometric twitches and two tetani (150 Hz) each separated by 1 min. For Figure 1P, the muscle was subjected to a force frequency protocol consisting of 6 stimulations of increasing frequency (1, 30, 50, 80, 100, and 150 Hz), one min apart. For results shown in Figure 1Q–T, muscles were incubated in a muscle strip myograph system (820MS, DMT, Denmark) at a 5–6 mN tension. Muscles were stimulated to contract using the following protocol: stimulus frequency 100 Hz; stimulus width 0.2 ms; stimulus train duration 1 sec train every 15 sec. The total stimulation protocol was 9 min.

Mice lacking OGT in skeletal muscle exhibit reduced fat mass but normal skeletal muscle morphology and contractility. (A) O-GlcNAc levels in human skeletal muscles of lean, obese, and type 2 diabetic individuals at basal (B) and after a hyperinsulinemic euglycemic clamp (Insulin (I)). Lane intensities were quantified between 15 and 300 kDa. Sample size of 8–10 people per group. Data are mean ± SEM and analyzed by two-way repeated measures ANOVA; ** indicates p < 0.01. (B) OGT, (C) O-GlcNAc, and (D) OGA immunoblotted in tibialis anterior (TA), soleus, and extensor digitorum longus (EDL) muscles from WT and mKO mice (n = 4). (E) Body weights of WT and mKO mice measured at 6, 9, 12, 16, and 30 weeks of age (n = 6–10). (F–H) Body composition: fat percentage (F), lean percentage (G), and muscle weights (H) of TA, soleus, and EDL in 16-week-old WT and mKO mice (n = 10). (I) Images comparing abdominal, inguinal (IngW) and epididymal (EpiW) white adipose tissue (WAT) depots from WT and mKO mice. (J) Quantification of adipose tissue weights expressed as percentage (%) body weight (n = 10). (K) Representative micrographs of H&E stained sections from epididymal (EpiW) and inguinal (IngW) white adipose tissues (WAT) of WT and mKO mice. Scale bar, 100 μm. The relative size of adipocytes in the EpiW and IngW of mKO mice was expressed as a percentage of WT (n = 10). (L) Immunoreactive uncoupling protein 1 (UCP1) in IngW of WT (left) and mKO (right) mice. Scale bar, 100 μm. (M–O) EDL muscle single-twitch contraction force (M), time-to-half relaxation tension (T1/2R; n = 10) (N), and time to peak of tension (n = 10) (O). (P) EDL muscle contractile force in response to increased stimulation frequency (n = 10). Data normalized to force at 150 Hz. (Q,R) First force train at 100Hz, 1 sec duration in soleus (Q; n = 5), and fatigue-ability measured as force produced over consecutive force trains at 100 Hz, 1 sec duration in soleus (R; n = 5). (S,T) First force train at 100 Hz, 1 sec duration in EDL (S; n = 5), and fatigue-ability measured as force produced over consecutive force trains at 100 Hz, 1 sec duration in EDL (T; n = 5). Data in B-T are means ± SEM from 16-week-old male mice. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice.