Suzette L. Pereira

Effect of β-hydroxy-β-methylbutyrate (HMB) on lean body mass during 10 days of bed rest in older adults

BACKGROUNDnLoss of muscle mass due to prolonged bed rest decreases functional capacity and increases hospital morbidity and mortality in older adults.nnnOBJECTIVEnTo determine if HMB, a leucine metabolite, is capable of attenuating muscle decline in healthy older adults during complete bed rest.nnnDESIGNnA randomized, controlled, double-blinded, parallel-group design study was carried out in 24 healthy (SPPB ≥ 9) older adult subjects (20 women, 4 men), confined to complete bed rest for ten days, followed by resistance training rehabilitation for eight weeks. Subjects in the experimental group were treated with HMB (calcium salt, 1.5 g twice daily - total 3 g/day). Control subjects were treated with an inactive placebo powder. Treatments were provided starting 5 days prior to bed rest till the end rehabilitation phase. DXA was used to measure body composition.nnnRESULTSnNineteen eligible older adults (BMI: 21-33; age: 60-76 year) were evaluable at the end of the bed rest period (Control n = 8; Ca-HMB n = 11). Bed rest caused a significant decrease in total lean body mass (LBM) (2.05 ± 0.66 kg; p = 0.02, paired t-test) in the Control group. With the exclusion of one subject, treatment with HMB prevented the decline in LBM over bed rest -0.17 ± 0.19 kg; p = 0.23, paired t-test). There was a statistically significant difference between treatment groups for change in LBM over bed rest (p = 0.02, ANOVA). Sub-analysis on female subjects (Control = 7, HMB = 8) also revealed a significant difference in change in LBM over bed rest between treatment groups (p = 0.04, ANOVA). However, differences in function parameters could not be observed, probably due to the sample size of the study.nnnCONCLUSIONSnIn healthy older adults, HMB supplementation preserves muscle mass during 10 days of bed rest. These results need to be confirmed in a larger trial.

β-Hydroxy-β-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMBs ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.

β-Hydroxy-β-methylbutyrate (HMB) enhances the proliferation of satellite cells in fast muscles of aged rats during recovery from disuse atrophy

Loss of myonuclei by apoptosis is thought to contribute to sarcopenia. We have previously shown, that the leucine metabolite, β-hydroxy-β-methylbutyrate (HMB) suppresses apoptotic signaling and the apoptotic index (the ratio of apoptotic positive to apoptotic negative myonuclei) during muscle disuse and during reloading periods after disuse in aged rats. However, it was not clear if the apoptotic signaling indexes were due only to preservation of myonuclei or if perhaps the total myogenic pool increased as a result of HMB-mediated satellite cell proliferation as this would have also reduced the apoptotic index. In this study, we tested the hypothesis that HMB would augment myogenic cells (satellite cells) proliferation during muscle recovery (growth) after a period of disuse in senescent animals. The hindlimb muscles of 34 month old Fisher 344 × Brown Norway rats were unloaded for 14 days by hindlimb suspension (HLS), and then reloaded for 14 days. The rats received either Ca-HMB (340 mg/kg body weight; n = 16), or the vehicle (n = 10) by gavage throughout the experimental period. HMB prevented the functional decline in maximal plantar flexion isometric force production during the reloading period, but not during HLS. HMB-treatment enhanced the proliferation of muscle stem cells as shown by a greater percentage of satellite cells that had proliferated (more BrdU positive, Pax-7 positive, and more Pax7/Ki67 positive nuclei) and as a result, more differentiated stem cells were present (more MyoD/myogenin positive myonuclei), relative to total myonuclei, in reloaded plantaris muscles as compared to reloaded muscles from vehicle-treated animals. Furthermore HMB increased the nuclear protein abundance of proliferation markers, inhibitor of differentiation-2 and cyclin A, as compared to vehicle treatment in reloaded muscles. Although HMB increased phosphorylated Akt during reloading, other mTOR related proteins were not altered by HMB treatment. These data show that HMB improved the proliferation of muscle stem cells in fast twitch plantaris muscles. Enhanced satellite cell proliferation leading to increased differentiated myonuclei should increase the transcriptional potential to support muscle hypertrophic changes and functional changes in sarcopenic muscles, and this could partly explain the reduced apoptotic index in HMB treated muscles. Indeed, muscle mass and fiber cross-sectional area were significantly greater in plantaris muscles from HMB-treated animal muscles after reloading as compared to vehicle-treated animals.

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-3-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 suspension (HLS) in senescent animals. Fischer 344 × Brown Norway inbred rats (age 34 months) 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<0.05) 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 μm(2)) vs. vehicle treated animals (1953.0±41.9 μm(2)). 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%) was 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.

Attenuation of muscle wasting in murine C2C12 myotubes by epigallocatechin-3-gallate

BackgroundLoss 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.MethodsStudies were conducted in vitro using murine C2C12 myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α).ResultsEGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10xa0μM. 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.ConclusionThe 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.

Green tea extract attenuates muscle loss and improves muscle function during disuse, but fails to improve muscle recovery following unloading in aged rats

In this study we tested the hypothesis that green tea extract (GTE) would improve muscle recovery after reloading following disuse. Aged (32 mo) Fischer 344 Brown Norway rats were randomly assigned to receive either 14 days of hindlimb suspension (HLS) or 14 days of HLS followed by normal ambulatory function for 14 days (recovery). Additional animals served as cage controls. The rats were given GTE (50 mg/kg body wt) or water (vehicle) by gavage 7 days before and throughout the experimental periods. Compared with vehicle treatment, GTE significantly attenuated the loss of hindlimb plantaris muscle mass (-24.8% vs. -10.7%, P < 0.05) and tetanic force (-43.7% vs. -25.9%, P <0.05) during HLS. Although GTE failed to further improve recovery of muscle function or mass compared with vehicle treatment, animals given green tea via gavage maintained the lower losses of muscle mass that were found during HLS (-25.2% vs. -16.0%, P < 0.05) and force (-45.7 vs. -34.4%, P < 0.05) after the reloading periods. In addition, compared with vehicle treatment, GTE attenuated muscle fiber cross-sectional area loss in both plantaris (-39.9% vs. -23.9%, P < 0.05) and soleus (-37.2% vs. -17.6%) muscles after HLS. This green tea-induced difference was not transient but was maintained over the reloading period for plantaris (-45.6% vs. -21.5%, P <0.05) and soleus muscle fiber cross-sectional area (-38.7% vs. -10.9%, P <0.05). GTE increased satellite cell proliferation and differentiation in plantaris and soleus muscles during recovery from HLS compared with vehicle-treated muscles and decreased oxidative stress and abundance of the Bcl-2-associated X protein (Bax), yet this did not further improve muscle recovery in reloaded muscles. These data suggest that muscle recovery following disuse in aging is complex. Although satellite cell proliferation and differentiation are critical for muscle repair to occur, green tea-induced changes in satellite cell number is by itself insufficient to improve muscle recovery following a period of atrophy in old rats.

Metabolomic profiling reveals severe skeletal muscle group-specific perturbations of metabolism in aged FBN rats

Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344xa0×xa0Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32xa0months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

Age associated decline in the conversion of leucine to β-Hydroxy-β-Methylbutyrate in rats.

BACKGROUNDnThe loss of muscle mass is considered to be a major factor contributing to strength decline during aging. β-Hydroxy-β-Methylbutyrate (HMB), a metabolite of leucine has been shown to enhance muscle protein synthesis and attenuate loss of muscle mass by multiple pathways. However, the production and regulation of endogenous levels of HMB over the lifespan have not been investigated.nnnOBJECTIVEnThe objective of the present study was to do a cross-sectional analysis of the basal plasma levels of HMB in male Sprague-Dawley rats of different ages and to compare the efficiency of conversion of leucine to HMB in young versus older rats.nnnMETHODSnPlasma levels of HMB and α-ketoisocaproate (KIC) were analyzed in rats of different age groups (3, 9, 12 and 24months old, n=10 per group). Levels of 4-HPPD, the enzyme involved in the conversion of KIC to HMB in the liver were determined by ELISA. The conversion efficiency of leucine to HMB was compared between 3 and 24month rats after an oral bolus dose of leucine.nnnRESULTSnEndogenous circulating levels of HMB were significantly reduced in older age rats compared to young rats (100±3.7 vs 156±10 (mean±SEM), ng/mL, p<0.001). A significant negative correlation was seen between HMB levels and age. The liver levels of 4-HPPD were found to be significantly lower in old versus young rats. Consistent with this, the conversion efficiency of leucine to HMB was significantly lower in the aged versus young cohorts.nnnCONCLUSIONSnIn summary, this study depicts for the first time that the basal levels of HMB, a metabolite of amino acid leucine, declines with age, and that this decline is due to perturbations in the key enzyme 4-HPPD which catalyzes the conversion of KIC to HMB. As a consequence, the efficiency of conversion of leucine to HMB is diminished in older rats compared to younger rats.

Epigallocatechin-3-gallate increases autophagy signaling in resting and unloaded plantaris muscles but selectively suppresses autophagy protein abundance in reloaded muscles of aged rats

ABSTRACT We have previously found that Epigallocatechin‐3‐gallate (EGCg), an abundant catechin in green tea, reduced apoptotic signaling and improved muscle recovery in response to reloading after hindlimb suspension (HS). In this study, we investigated if EGCg altered autophagy signaling in skeletal muscle of old rats in response to HS or reloading after HS. Fischer 344 × Brown Norway inbred rats (age 34 months) were given 1 ml/day of purified EGCg (50 mg/kg body weight), or the same sample volume of the vehicle by gavage. One group of animals received HS for 14 days and the second group of rats received 14 days of HS, then the HS was removed and they were allowed to recover by ambulating normally around the cage for two weeks. EGCg decreased a small number of autophagy genes in control muscles, but it increased the expression of other autophagy genes (e.g., ATG16L2, SNCA, TM9SF1, Pink1, PIM‐2) and HS did not attenuate these increases. HS increased Beclin1, ATG7 and LC3‐II/I protein abundance in hindlimb muscles. Relative to vehicle treatment, EGCg treatment had greater ATG12 protein abundance (35.8%, P < 0.05), but decreased Beclin1 protein levels (− 101.1%, P < 0.05) after HS. However, in reloaded muscles, EGCg suppressed Beclin1 and LC3‐II/I protein abundance as compared to vehicle treated muscles. EGCg appeared to “prime” autophagy signaling before and enhance autophagy gene expression and protein levels during unloading in muscles of aged rats, perhaps to improve the clearance of damaged organelles. However, EGCg suppressed autophagy signaling after reloading, potentially to increase the recovery of hindlimb muscles mass and function after loading is restored. HIGHLIGHTSEpigallocatechin‐3‐gallate (EGCg) supplementation increased autophagy expression in aged muscles of control rodents.EGCg treatment maintained muscle autophagy signaling at high levels in during muscle disuse in aging.EGCg suppressed autophagy protein abundance when muscles were reloaded after disuse in aging.EGCg treatment primed autophagy before unloading and suppressed muscle autophagy signaling after reloading.

Green Tea Supplementation: Current Research, Literature Gaps, and Product Safety

Green Tea Supplementation: Current Research, Literature Gaps, and Product Safety nThis review broadly addresses the impacts of green tea (GT) and its extracts in a number of clinically-important areas, as listed below. It focuses on the available human research, and randomized, controlled trials, where possible. GT’s effects are not well established, due to the prevalence of conflicting data. It does appear that if GT is to have a positive effect on these outcomes, it likely needs to be in combination with caffeine.