Figure 1.
The horizontal arrows show the Chronic Low Frequency Stimulation period 1 hour (A) or 24 hours (B) after recovering from the surgery (4 days). Then both stimulated and non-stimulated contralateral tibialis anterior muscles were sampled at 0 hours (right after CLFS), 1, 6, 12 and 24 hours of rest. Collection of the samples was done always at the same hour of the day for both protocols.
Figure 2.
Glycogen content and the absorbance branching index.
(A) Glycogen content of 1 hour (▪ bar) or 24 hours (▪ bar) stimulated muscles (µmols of glucose/g tissue). (B) Wavelength of the maximum absorbance peak (nm) as branching index (▪ dots are 1 hour CLFS samples and ▪ dots are 24 hours CLFS samples). Control group (□ bar or dot) was composed with the pool of all contralateral non-stimulated muscle samples results (n = 50). n = 5 for the stimulated and rested groups. (C) The same amount (2.4 micrograms) of control (white circles), poorly branched normal content (24 h CLFS+1 h rest, grey triangles), poorly branched supercompensated (24 h CLFS+6 h rest, grey diamond) and normal branched supercompensated (black squares, 24 h CLFS+24 h rest) purified glycogen samples were elongated using GPh-b. Glucose incorporation from G1P was monitored at 5, 15, 30, 4 and 60 minutes. n = 3–5. Data are means ± SE. Bars or dots with the same letter are not significantly different from each other. *: p<0.05 compared with control.
Table 1.
Blood parameters, metabolites and nucleotide levels in muscle.
Figure 3.
Fold change in hk2 mRNA content of 1 hour (▪) or 24 hours (▪) stimulated samples compared to control situation measured with Real-Time PCR. Control group (□) was composed with the pool of all contralateral non-stimulated muscle samples results (n = 50). n = 5 for all the other groups. Data are means ± SE. Bars with the same letter are not significantly different from each other.
Table 2.
Enzyme activities in skeletal muscle.
Figure 4.
Change of glycogenin-1 protein content.
Fold change in glycogenin normalized by total protein content of 1 hour (▪) or 24 hours (▪) stimulated samples compared to control situation (□) measured by Western-blot of Glycogenin-1. The inset shows a representative blot for each situation (from 0 to 24 hours of rest): in the upper panel samples with 1 hour of CLFS, and the lower panel the groups with 24 hours of CLFS. C is the contralateral non-stimulated control sample of each sample showed. In the bar chart the control group was composed with the pool of all contralateral1 non-stimulated muscle samples results (n = 50). n = 5 for the stimulated and rested groups. Data are means ± SE. There was no statistical difference under any condition.
Figure 5.
Total mGS protein and phosphorylation content of the different sites.
Fold change of 1 hour (▪) or 24 hours (▪) stimulated samples compared to control situation (□) measured by Western-blot of: (A) Total mGS protein content normalized by total protein and phosphorylation of mGS in (B) site 1a, (C) site 1b, (D) site 2, (E) site 2+2a, (F) site 3a and (G) site 3b, normalized by mGS. The control was composed with the pool of all contralateral non-stimulated muscle samples results (n = 50). For the stimulated and rested groups n = 5. The upper panels of each chart contain representative blots to the total mGS or the phosphorylated sites: 1 hour CLFS the upper lane and 24 hours CLFS the lower lane. C: The correspondent non-stimulated control, and 0 h, 1 h, 6 h, 12 h and 24 h are the time of rest after the stimulation. Data are expressed as box and whisker plot with the median as a transversal line in the box. Groups with the same letter are not significantly different from each other.
Figure 6.
(A) Representative blots of AMPK-P (T172), AMPKα1 and AMPKα2 isoforms. 1 hour CLFS samples on the left and 24 hours CLFS on the right. Samples are C: The correspondent non-stimulated control, and 0 h, 1 h, 6 h, 12 h and 24 h are the time of rest after the stimulation. (B) Quantification of the AMPK-P (T172) Western-blot blot normalized by AMPKα1 in fold change (from 0 to 160 fold) compared to control and in a different scale (C) with a break from 7 to 35 to better display the data in the control samples and samples rested from 6 to 24 hours. Quantification of AMPKα1 (D) and AMPKα2 (E) in fold change compared to control from the Western-blot normalized by total protein content. 1 hour (▪) and 24 hours (▪) stimulated samples n = 5. Control group (□) was composed with the pool of all contralateral non-stimulated muscle samples results (n = 50). Data are means ± SE. Bars with the same letter are not significantly different from each other.