Attenuation of muscle wasting in murine C2C12myotubes by epigallocatechin-3-gallate

Background: Loss of muscle protein is a common feature of wasting diseases where currently treatment is limited. This study investigates the potential of epigallocatechin-3-gallate (EGCg), the most abundant catechin in green tea, to reverse the increased protein degradation and rescue the decreased protein synthesis which leads to muscle atrophy. Methods: Studies were conducted in vitro using murine C2C12myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α). Results: EGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10 μM. Serum starvation increased expression of the proteasome 20S and 19S subunits, as well as the proteasome ‘chymotrypsin-like’ enzyme activity, and these were all attenuated down to basal values in the presence of EGCg. Serum starvation did not increase expression of the ubiquitin ligases MuRF1 and MAFbx, but EGCg reduced their expression below basal levels, possibly due to an increased expression of phospho Akt (pAkt) and phospho forkhead box O3a (pFoxO3a). Attenuation of protein degradation by EGCg was increased in the presence of ZnSO4, suggesting an EGCg-Zn2+complex may be the active species. Conclusion: The ability of EGCg to attenuate depressed protein synthesis and increase protein degradation in the myotubule model system suggests that it may be effective in preserving skeletal muscle mass in catabolic conditions

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

Epigallocatechin- 3-gallate improves plantaris muscle recovery after disuse in aged rats

Aging exacerbates muscle loss and slows the recovery of muscle mass and function after disuse. In this study we investigated the potential that epigallocatechin gallate (EGCg), an abundant catechin in green tea, would reduce signaling for apoptosis and promote skeletal muscle recovery in the fast plantaris muscle and the slow soleus muscle after hindlimb unloading (HLS) in senescent animals. Fischer 344 × Brown Norway inbred rats (age 34 mo.) received either EGCg (50 mg/kg body weight), or water daily by gavage. One group of animals received HLS for 14 days and a second group of rats received 14 days of HLS, then the HLS was removed and they recovered from this forced disuse for 2 weeks. Animals that received EGCg over the HLS followed by 14 days of recovery, had a 14% greater plantaris muscle weight (p \u3c0.05) as compared to the animals treated with the vehicle over this same period. Plantaris fiber area was greater after recovery in EGCg (2715.2 ± 113.8 μm2) vs. vehicle treated animals (1953.0 ± 41.9 μm2). In addition, activation of myogenic progenitor cells was improved with EGCg over vehicle treatment (7.5% vs. 6.2%) in the recovery animals. Compared to vehicle treatment, the apoptotic index was lower (0.24% vs. 0.52%), and the abundance of pro-apoptotic proteins Bax (−22%), and FADD (−77%) were lower in EGCg treated plantaris muscles after recovery. While EGCg did not prevent unloading-induced atrophy, it improved muscle recovery after the atrophic stimulus in fast plantaris muscles. However, this effect was muscle specific because EGCg had no major impact in reversing HLS-induced atrophy in the slow soleus muscle of old rats

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice.

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice

HMB enhances C₂C₁₂ myoblasts cell viability and proliferation.

<p>C₂C₁₂ myoblasts were treated with either 25 μM or 125 μM free acid HMB or vehicle control. Cell viability was assessed at 48 and 72 hours proliferation using the Trypan Blue exclusion assay. Experiments were performed under conditions of both low serum (A) and normal serum (B), (Control, n = 6; 25 μM HMB, n = 3; 125 μM HMB, n = 3, * denotes significant difference compared to control: 3% FBS, P = 0.044; 10% FBS, P = 0.031, One-way ANOVA). C) C₂C₁₂ myoblasts were treated with 25 μM or125 μM HMB or vehicle control for 48 hours in proliferation media (10% FBS) at which point total viable cells were counted while gating out cellular debris with the Scepter™ Automated Cell Counter (Control, n = 6; 25 μM HMB, n = 3; 125μM HMB, n = 3,* denotes significance compared to control: 25 μM HMB, P<0.042; 125 μM HMB, P<0.01, One-way ANOVA).</p

Cellular and Physiological Effects of Dietary Supplementation with <i>β</i>-Hydroxy-<i>β</i>-Methylbutyrate (HMB) and <i>β</i>-Alanine in Late Middle-Aged Mice

<div><p>There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C₂C₁₂ myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid <i>β</i>-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C₂C₁₂ myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C₂C₁₂ myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an <i>ex vivo</i> muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with <i>β</i>-alanine, or diet with HMB and <i>β</i>-alanine. In <i>β</i>-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus <i>β</i>-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm², P = 0.021). Also in EDL muscle, <i>β</i>-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and <i>β</i>-alanine supplementation on skeletal muscle function in aging mice.</p></div

Kinetic properties of individual contractions from EDL and soleus muscles.

<p>A) Rate of force generation of individual contractions from EDL muscle at the stimulatory frequencies ranging from 1–130 Hz (^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control diet. The bracket indicates significance at all stimulatory frequencies tested). B) The time to reach peak contractile force in contractions from EDL muscle stimulated with the frequencies of 1–130 Hz (* denotes significant difference in HMB diet compared to control diet). C) The time constant (tau) in the decaying exponential fit to the tail of the contractions of EDL muscle stimulated at 1–130 Hz. D) Rate of force generation of individual contractions from soleus muscle at the stimulatory frequencies ranging from 1–130 Hz (^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control. E) The time to reach peak contractile force in contractions from soleus muscle stimulated with the frequencies of 1–130 Hz (* denotes significant difference in HMB diet compared to control diet, ^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control, ^<b>b</b> denotes significant difference in HMB + <i>β</i>-alanine group compared to control). F) The time constant (tau) in the decaying exponential fit to the tail of the contractions of soleus muscle stimulated at 1–130 Hz. (EDL: Control-n = 24 muscles, HMB-n = 23 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles. SOL: Control-n = 24 muscles, HMB-n = 24 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles).</p

EDL and soleus muscle optimal length and mass.

<p>EDL and soleus muscle optimal length and mass.</p

Fatiguing stimulation and recovery from fatigue in EDL and soleus muscles.

<p>Alternating frequencies of 80 Hz and 20 Hz were used to fatigue the muscles intermittently for 5 minutes, with a periodicity of one second. The periodicity, or interval between stimulations, was then extended to one minute to allow force recovery in the absence and presence of 5 mM caffeine. A and B) EDL fatigue and recovery, respectively, using the low stimulatory frequency of 20 Hz. D) EDL fatigue and recovery, respectively using the high frequency of 80 Hz. E and F) Soleus fatigue and recovery, respectively, using the low stimulatory frequency of 20 Hz (<b>@</b> denotes P = 0.049). G and H) Soleus fatigue and recovery, respectively, using the high frequency of 80 Hz. (EDL: Control-n = 22 muscles, HMB-n = 23 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles. SOL: Control-n = 22 muscles, HMB-n = 24 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 22 muscles).</p

Summary of <i>in vivo</i> study timeline and muscle contractility assay.

<p>A) Schematic representation of the dietary intervention study performed on 48 C57BL/6nTac male mice. All mice were 19 months old at the time of sacrifice for contraction studies. B) Representative tracing of force data from the <i>ex vivo</i> contractility assay obtained from one muscle (<i>X-axis</i>: Time; <i>Y-axis</i>: Force). Equilibration, fatigue, and recovery from fatigue protocols are performed using alternating high (80 Hz) and low (20 Hz) frequencies of stimulation. <i>Inset</i>: Magnified view of fatiguing stimulations using alternating high (80 Hz) and low (20 Hz) stimulation frequencies.</p