Table 1.
Muscle and body Weights.
Table 2.
Muscle function was increased by LFES.
Fig 1.
Acu-LFES prevents diabetes-induced muscle fiber cross-sectional area (MCS) decrease.
A representative cross-sectional area of EDL muscle from normal control (control), Acu-LFES treated normal controls (Acu-LFES), diabetes or diabetes/Acu-LFES mice are showed (A). The cryosections of EDL muscle were immunostained with anti-laminin antibody. The First bar graph (B) shows the average size of myofibers determined from six mice x 8 sections/mouse/group (Bars: mean ± s.e.; n = 9/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes). The frequency distribution of fiber cross-sectional area in control (grey bar), diabetes (open bar) and diabetes/Acu-LFES (black bar) mice is presented as percent fibers/size of fibers (C).
Fig 2.
Acu-LFES increases muscle regeneration of mRNA markers.
Pax7, myoD, myogenin and eMyHC were measured by conventional RT-PCR in combined gastrocnemius and EDL muscle lysates from control, Acu-LFES, diabetes or diabetes/Acu-LFES mice. The bar graph compares the densities of mRNA bands in each group expressed as a fold-change from levels in control mice which is represented by a line at 1-fold. All band densities were normalized to the density of the 18S rRNA band (Bars: mean ± s.e.; n = 12/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).
Fig 3.
Acu-LFES counteracts diabetes-induced decrease of muscle regeneration proteins.
Muscle proteins lysates were prepared from combined gastrocnemius and EDL muscles from control, Acu-LFES, diabetes or diabetes/Acu-LFES mice. Muscle regeneration related proteins (Pax7, myoD, myogenin, eMyHC) and GAPDH were measured by western blotting. The bar graph compares the protein band densities in each treatment group expressed as a fold-change from levels in control mice (represented by a line at 1-fold). All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n = 12/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).
Fig 4.
Acu-LFES increase satellite cells migration in normal and diabetic mice.
A representative cross-sections from EDL muscles after staining for laminin (green) and counterstaining with DAPI (blue) are shown in control, Acu-LFES, diabetes or diabetes/Acu-LFES mice. The white arrows point to central nuclei inside of myofibers which indicates satellite cells migration. The bar graph shows the nuclei number inside (central nuclei) per 500 muscle fibers (Bars: mean ± s.e.; n = 9; # = p<0.05 vs. control).
Fig 5.
Acu-LFES improves protein synthesis markers in skeletal muscle of diabetic mice.
The protein synthesis related markers mTOR, p-mTOR, p70S6K and p-p70S6K were measured by western blotting in combined gastrocnemius and EDL muscle lysates from control, Acu-LFES, diabetes or diabetes/Acu-LFES mice. The bar graph compares the densities of protein bands in each group expressed as a fold-change from levels in control mice which is represented by a line at 1-fold. All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n = 12/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).
Fig 6.
Acu-LFES improve Akt phosphorylation and inhibit FoxO activation in the muscle of diabetic mice.
The protein metabolism related proteins Akt, p-Akt, FoxO1 and p-FoxO1 were measured by western blotting in combined gastrocnemius and EDL muscle lysates from control, Acu-LFES, diabetes or diabetes/Acu-LFES mice. The bar graph compares the densities of protein bands in each group expressed as a fold-change from levels in control mice which is represented by a line at 1-fold. All band densities were normalized to the density of GAPDH (Bars: mean ± s.e.; n = 12/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).
Fig 7.
Acu-LFES upregulates IGF-1 mRNA and protein in the muscle of diabetic mice.
Panel A: Total RNA isolated from combined gastrocnemius and EDL muscles of control, Acu-LFES, diabetes or diabetes/Acu-LFES mice were assayed for IGF-1 expression by real time qPCR. The bar graph shows mRNA from the muscles of each group of mice. Results are normalized to 18S RNA (Bars: mean ± s.e.; n = 9/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes). Panel B: IGF-1 protein levels were measured by ELISA in in combined gastrocnemius and EDL lysates from control, Acu-LFES treated normal controls (Acu-LFES), diabetes or diabetes/Acu-LFES mice. The bar graph shows the IGF-1 protein levels in each group. IGF-1 levels in the muscle lysates were normalized to the total protein concentration (Bars: mean ± s.e.; n = 9; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).
Fig 8.
Acu-LFES increase miR-1 and miR-206 microRNA in the muscle of control and diabetic mice.
Total RNA was isolated from combined gastrocnemius and EDL muscles of control, Acu-LFES, diabetes or diabetes/Acu-LFES, and then assayed for specific microRNA expression. miR-1 (A), miR-206 (B), miR-133a (C) and miR-133b (D) expressions were measured using real time qPCR with LNA-enhanced oligonucleotide primers. The bar graph shows microRNA levels in each group expressed as a fold-change from levels in control mice which is represented by a line at 1-fold. All band densities were normalized to the density of U6 RNA (Bars: mean ± s.e.; n = 6/group; * = p<0.05 vs. control and # = p<0.05 vs. diabetes).