β-Hydroxy-β-Methylbutyrate (HMB) Normalizes Dexamethasone-Induced Autophagy-Lysosomal Pathway in Skeletal Muscle

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.This project has been funded by Abbott Nutrition R&D

Effects of HMB supplementation on morphometric analysis and hind limb strength in DEX-treated rats.

<p><b>(A)</b> Muscle fiber cross-sectional area (μm²). Measurements were made 21 days post treatment. <b>(B)</b> Hind limb strength in animals submitted to saline, DEX or HMB/DEX. Values are expressed as mean ± SEM (n = 7). ^ap<0.05 compared with Control group. ^bp<0.05 compared with DEX group.</p

Effects of HMB on Ub-C promoter activity and expression of Atrogin-1 and MuRF1 induced by DEX.

<p><b>(A)</b> Cells were transiently transfected with an UbC-luciferase reported plasmid and differentiated into myotubes before treatments to evaluate UbC gene transcription (n = 6). Western blots for Atrogin-1 <b>(B)</b> and MuRF1 <b>(D)</b>. Densitometric quantification of protein abundance using actin as a reference control is shown. Signal densities from untreated cells were assigned a value of 100% (n = 4). mRNA levels for Atrogin-1 <b>(C)</b> and MuRF1 <b>(E)</b> were measured in samples from all the experimental groups (n = 8). Results are expressed as means ± SEM. ^ap ‹ 0.05 compared with untreated cells and ^bp ‹ 0.05 compared with DEX-treated cells.</p

Effects of HMB supplementation on body weight, lean body mass and muscle weight in DEX-treated rats.

<p><b>(A)</b> Body weight and <b>(B)</b> lean body mass analysis of each group (Control, DEX and HMB/DEX). Day 0 was considered as the day of initiation of dexamethasone treatment. Wet weight (grams) of soleus <b>(C)</b> and gastrocnemius <b>(D)</b> for each group at the end of the experiment. Values are expressed as mean ± SEM (n = 7). ^ap<0.05 compared with Control group. ^bp<0.05 compared with DEX group.</p

Effects of HMB supplementation on the autophagosome formation induced by DEX.

<p>Autophagosomes were quantified by counting GFP-LC3 punctae/cell of at least 4 fields per treatment. Scale bar in the microphotographs corresponds to 25 μm. Results represent means ± SEM (n = 4).^a p < 0.05 compared with untreated control cells; ^b p < 0.05 compared with DEX-treated cells.</p

Effects of HMB supplementation on the regulation of LC3, p62, Bnip3 and S6K1 expression induced by DEX.

<p><b>(A)</b> Immunoblotting for LC3 in absence or presence of Bafilomycin A1. Two bands are shown, corresponding to LC3-I and the lipidated form LC3-II. <b>(B)</b> Levels of p62 were evaluated by immunoblot analysis in the absence or presence of 100 nM Bafilomycin A1. <b>(C)</b> mRNA levels for Bnip3 were measured by quantitative real-time PCR analysis in samples from all the experimental groups (n = 8). <b>(D)</b> Phosphorylation level of S6K1. Signal densities from untreated cells were assigned a value of 100%. Results are expressed as means ± SEM (n = 4). ^a p ‹ 0.05 compared with untreated cells and ^b p ‹ 0.05 compared with DEX-treated cells.</p

Effects of HMB on the phosphorylation of FoxO3a, Akt and ERK1/2 and on FoxO transcriptional activity in presence of DEX.

<p>Cells were lysed and total protein was immunoblotted with specific phospho- and total-antibodies against FoxO3 (<b>A),</b> Akt/PKB (<b>B</b>) or ERK1/2 <b>(C)</b>. Signal densities from untreated cells were assigned a value of 100%. Results are expressed as means ± SEM (n = 4).^a p ‹ 0.05 compared with untreated cells and ^b p ‹ 0.05 compared with DEX- treated cells. <b>(D)</b> Cells were transiently transfected with a FoxO luciferase reported plasmid and differentiated into myotubes before treatments to evaluate FoxO-dependent transcription. Cells were pre-incubated for 30 min with 10 μM PD98059 and 20 μM LY294002, then incubated with 25 μM HMB for 48 h and finally incubated with 5 μM DEX in the presence or absence of effectors. Inhibitors were maintained during the experiment. Results are expressed as means ± SEM (n = 6). ^ap ‹ 0.05 compared with control cells in the presence of each inhibitor, ^bp ‹ 0.05 compared with DEX- treated cells in the presence of each inhibitor and ^cp ‹ 0.05 compared with control cells in the absence of inhibitors.</p

Effect of HMB on protein degradation and synthesis in rat L6 myotubes treated with DEX.

<p>Results are shown as the increase in protein degradation and the decrease in protein synthesis (%) due to the presence of DEX when compared with basal levels (non-treated cells). Results are expressed as means ± SEM (n = 8).^ap ‹ 0.05 compared with DEX treated cells.</p