Leucine supplementation improves skeletal muscle regeneration after cryolesion in rats

This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.FAPESP, 2010/52520-0FAPESP, 2012/15276-

Leucine supplementation does not activate PI3K/Akt/mTOR signalling pathway in regenerating muscles.

<p><b>A</b>: Representative Western blots of elements from the PI3K/Akt/mTOR signalling pathway. Muscle groups are identified at the top. <i>C</i>, intact control muscle; <i>Leu</i>, control muscles supplemented only with leucine from the time point of post-cryolesion day 10; <i>Cryo</i>, damaged muscle analysed on post-cryolesion day 10; and <i>Cryo+Leu</i>, leucine-supplemented damaged muscle analysed on post-cryolesion day 10. <b>B</b>: Densitometry analyses of elements from the PI3K/Akt/mTOR signalling pathway. Blots were reacted with antibodies specific for mTOR; phosphorylated mTOR at Ser²⁴⁴⁸ residue; phosphorylated p70 at Ser³⁷¹ and Thr³⁸⁹ residues; 4E-BP1; p-4E-BP1 at Thr⁷⁰ and Thr^37/46 residues; eIF4E; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as standard. Mw: molecular weight. Data are presented as mean±SD (n = 4). ^a<i>p</i><0.05 vs. C and Leu; ^b<i>p</i><0.05 vs. Cryo.</p

Soleus muscle weight and body weight of rats at post-cryolesion days 1, 3, and 10.

<p>Control: intact muscles; Leu: muscles supplemented with leucine; Cryo: cryolesioned muscles; Cryo+Leu: cryolesioned muscles supplemented with leucine;</p><p>Control/Cryo: animals that had the left soleus muscle cryolesioned and the right soleus muscles used as control; Leu/Cryo+Leu: animals that were supplemented with leucine and had the left soleus muscle cryolesioned and the right soleus muscles used as control. ^a<i>p</i><0.05 vs. Control and Leu in the same post-cryolesion time point; ^b<i>p</i><0.05 vs. matched group on Day 1 after cryolesion (analysis of variance followed by Tukey's post hoc test for multiple comparison.</p

Leucine supplementation reduces ubiquitinated protein accumulation.

<p><b>A</b>: Representative Western blots of ubiquitinated proteins. Samples were analysed at two different time points of experimental protocol (3 and 10 days post-cryolesion). Muscle groups and experimental time points are identified at the top. <i>C</i>, intact control muscle; <i>Leu</i>, control muscles supplemented only with leucine from the time points of post-cryolesion day 3 and 10, respectively; <i>Cryo</i>, damaged muscle analysed on post-cryolesion days 3 and 10, respectively; and <i>Cryo+Leu</i>, leucine-supplemented damaged muscle analysed on post-cryolesion days 3 and 10, respectively. <b>B</b>: Densitometry analyses of ubiquitinated proteins at 3 (panel <i>a</i>) and 10 days (panel <i>b</i>) of experimental protocol. Blots were reacted with antibody specific for ubiquitin. Mw: molecular weight. Data are presented as mean±SD (n = 4). ^a<i>p</i><0.05 vs. C and Leu; ^b<i>p</i><0.05 vs. Cryo.</p

Leucine supplementation reduces FOXO3a activation in regenerating muscles.

<p><b>A</b>: Soleus muscle cross sections immunostained for FOXO3a. Activation of FOXO3a was assessed by quantifying FOXO3a positive nuclei per cubic millimeter. Post-cryolesion times are identified at the top. <i>C</i>: intact control muscle; <i>Leu</i>: control muscles supplemented only with leucine from the time points of post-cryolesion day 3 and 10, respectively; <i>Cryo</i>: damaged muscle analysed on post-cryolesion days 3 or 10, respectively; and, <i>Cryo+Leu</i>: leucine-supplemented damaged muscle analysed on post-cryolesion days 3 or 10, respectively. Arrowheads indicate FOXO3a, DAPI (nucleus staining), and merge of FOXO3a and DAPI. Bar = 50 µm. <b>B</b>: Number of FOXO3a positive nuclei per cubic millimeter of soleus muscles from post-cryolesion day 3 (panel <i>a</i>) and 10 days (panel <i>b</i>). Data are presented as mean±SD (n = 5). ^a<i>p</i><0.05 vs. C; ^b<i>p</i><0.05 vs. Cryo group.</p

Leucine supplementation reduces area density of collagen type III in regenerating muscles.

<p><b>A</b>: Soleus muscle cross sections immunostained for collagen type III. <i>C</i>: intact control muscle; <i>Leu</i>: control muscles supplemented only with leucine from the time point of post-cryolesion day 10; <i>Cryo</i>: damaged muscle analysed on post-cryolesion day 10; and, <i>Cryo+Leu</i>: leucine-supplemented damaged muscle analysed on post-cryolesion day 10. Bar = 50 µm. <b>B</b>: Area density of collagen type III (percentage of the whole muscle cross section) in soleus muscles. Data are presented as mean±SD (n = 5). ^a<i>p</i><0.05 vs. C and Leu; ^b<i>p</i><0.05 vs. Cryo.</p

Leucine supplementation reduces incidence of macrophages.

<p>Number of macrophages per cubic millimeter of soleus muscles. <i>C</i>: intact control muscles; <i>Leu</i>: control muscles supplemented only with leucine from the time point of post-cryolesion day 3; <i>Cryo</i>: damaged muscles analysed on post-cryolesion day 3; and, <i>Cryo+Leu</i>: leucine-supplemented damaged muscles analysed on post-cryolesion day 3. Data are presented as mean±SD (n = 5). ^a<i>p</i><0.05 vs. C and Leu; ^b<i>p</i><0.05 vs. Cryo.</p