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Using a unilateral model of resistance training, we show for the first time that a protein-polyphenol beverage increases initial rates of myofibrillar protein synthesis and promotes early functional improvements. Following a prolonged period of training, this strategy also increases type II fiber hypertrophy and causes large individual variation in gains in quadricep muscle cross-sectional area.

The purpose of this study was to validate the efficacy of a customized vitamin-mineral supplement on blood biomarkers in pre-menopausal females. Women (21–40 years old) who were apparently healthy were recruited from the local community (ClinicalTrials.gov trial registration NCT03828097). Pretesting (PRE) occurred in the morning 5 ± 2 days following each participant's menses and involved a fasted blood draw, body mass assessment, and blood pressure assessment. Participants were then randomly assigned in a double-blinded fashion to either the multivitamins (MV) (n = 43) or placebo group (n = 51). Participants consumed two capsules per day with breakfast for 12 weeks. Following the trial, participants reported to the laboratory for POST assessments, which replicated PRE procedures. Red blood cell fatty acid and serum micronutrient analyses were performed in a blinded fashion at hematology laboratories. A group × time interaction was observed for serum vitamin D levels (p < 0.001). MV increased levels from PRE to POST (+43.7%, p < 0.001), whereas no change occurred in the placebo group. Additionally, 78% of MV participants at PRE exhibited inadequate vitamin D levels (p < 0.001). MV increased serum folate from PRE to POST (p < 0.001), whereas no change occurred in the placebo group. Red blood cell omega-3 fatty acid content increased from PRE to POST in the MV group (p < 0.001) and placebo group (p < 0.05), although POST values were greater in the MV group (p < 0.001). An interaction was observed for serum HDL cholesterol levels (p = 0.047), and a non-significant increase in this variable from PRE to POST occurred in the MV group (p = 0.060). Four-day food recalls indicated MV increased intake of omega-3 fatty acids, vitamin D, folate, and other micronutrients. In summary, MV supplementation increased serum vitamin D, serum folate, and red blood cell omega-3 fatty acid levels. However, these data are limited to healthy females, and more research is needed to examine if MV can affect metabolic disturbances in individuals with micronutrient deficiencies.

OBJECTIVES: In vitro dissolution tests are valuable first-step tools in the development of bioavailable delivery systems, making it possible to assess the performance of novel technologies in releasing active ingredients through the amount dissolved in a dissolution medium. The objective of this study was to evaluate whether phosphatidyl choline-enriched lipid encapsulation releases the majority of vitamin C as ascorbic acid past the stomach in a standard in vitro dissolution procedure and to assess the release profile. METHODS: A novel phosphatidyl choline-enriched lipid encapsulation that is solid at room temperature was tested for dissolution in a standard dissolution apparatus according to compendial United States Pharmacopeia methods, as per Good Manufacturing Practices for dietary supplements. One serving (included 1000 mg ascorbic acid) was placed into vessels containing simulated gastric fluid (0.1 M HCl) for 120 minutes then changed to simulated intestinal fluid (buffered 2% sodium lauryl sulfate, pH 6.8) for an additional 360 minutes. Aliquots were tested for ascorbic acid concentration at 8 time points by titration. The data was compared with 1000 mg of regular ascorbic acid in a capsule format. RESULTS: The phosphatidyl choline-enriched lipid encapsulation released 36% of the vitamin C at 2 hours in the acid phase and released 56% at 3 hours, 67% at 4 hours, 85% at 6 hours and 98% at 8 hours in the intestinal phase. Regular vitamin C filled capsules released 100% of the vitamin C at 30 minutes. CONCLUSIONS: The dissolution results indicated that phosphatidyl choline-enriched lipid encapsulation can pass the stomach and release the majority of vitamin C in the small intestine. The encapsulation demonstrated a sustained and prolonged release of vitamin C over an 8 hour period. The release profile observed in this in vitro study suggests phosphatidyl choline-enriched lipid encapsulation may improve nutrient absorption and bioavailability which requires further testing including human clinical trials. FUNDING SOURCES: This study was supported by Lonza (Greenwood, SC) and Ritual (Natals Inc, Los Angeles, CA).

The contribution of myofibrillar protein synthesis (MyoPS) to recovery from skeletal muscle damage in humans is unknown. Recreationally active men and women consumed a daily protein-polyphenol beverage targeted at increasing amino acid availability and reducing inflammation (PPB; n = 9), both known to affect MyoPS, or an isocaloric placebo (PLA; n = 9) during 168 h of recovery from 300 maximal unilateral eccentric contractions (EE). Muscle function was assessed daily. Muscle biopsies were collected for 24, 27, 36, 72, and 168 h for MyoPS measurements using (2)H(2)O and expression of 224 genes using RT-qPCR and pathway analysis. PPB improved recovery of muscle function, which was impaired for 5 days after EE in PLA (interaction P < 0.05). Acute postprandial MyoPS rates were unaffected by nutritional intervention (24–27 h). EE increased overnight (27–36 h) MyoPS versus the control leg (PLA: 33 ± 19%; PPB: 79 ± 25%; leg P < 0.01), and PPB tended to increase this further (interaction P = 0.06). Daily MyoPS rates were greater with PPB between 72 and 168 h after EE, albeit after function had recovered. Inflammatory and regenerative signaling pathways were dramatically upregulated and clustered after EE but were unaffected by nutritional intervention. These results suggest that accelerated recovery from EE is not explained by elevated MyoPS or suppression of inflammation. NEW & NOTEWORTHY The present study investigated the contribution of myofibrillar protein synthesis (MyoPS) and associated gene signaling to recovery from 300 muscle-damaging, eccentric contractions. Measured with (2)H(2)O, MyoPS rates were elevated during recovery and observed alongside expression of inflammatory and regenerative signaling pathways. A nutritional intervention accelerated recovery; however, MyoPS and gene signaling were unchanged compared with placebo. These data indicate that MyoPS and associated signaling do not explain accelerated recovery from muscle damage.

Context The early events regulating the remodeling program following skeletal muscle damage are poorly understood. Objective The objective of this study was to determine the association between myofibrillar protein synthesis (myoPS) and nuclear factor-kappa B (NF-κB) signaling by nutritionally accelerating the recovery of muscle function following damage. Design, Setting, Participants, and Interventions Healthy males and females consumed daily postexercise and prebed protein-polyphenol (PP; n = 9; 4 females) or isocaloric maltodextrin placebo (PLA; n = 9; 3 females) drinks (parallel design) 6 days before and 3 days after 300 unilateral eccentric contractions of the quadriceps during complete dietary control. Main Outcome Measures Muscle function was assessed daily, and skeletal muscle biopsies were taken after 24, 27, and 36 hours for measurements of myoPS rates using deuterated water, and gene ontology and NF-κB signaling analysis using a quantitative reverse transcription PCR (RT-qPCR) gene array. Results Eccentric contractions impaired muscle function for 48 hours in PLA intervention, but just for 24 hours in PP intervention (P = 0.047). Eccentric quadricep contractions increased myoPS compared with the control leg during postexercise (24–27 hours; 0.14 ± 0.01 vs 0.11 ± 0.01%·h-1, respectively; P = 0.075) and overnight periods (27–36 hours; 0.10 ± 0.01 vs 0.07 ± 0.01%·h-1, respectively; P = 0.020), but was not further increased by PP drinks (P > 0.05). Protein-polyphenol drinks decreased postexercise and overnight muscle IL1R1 (PLA = 2.8 ± 0.4, PP = 1.1 ± 0.4 and PLA = 1.9 ± 0.4, PP = 0.3 ± 0.4 log2 fold-change, respectively) and IL1RL1 (PLA = 4.9 ± 0.7, PP = 1.6 ± 0.8 and PLA = 3.7 ± 0.6, PP = 0.7 ± 0.7 log2 fold-change, respectively) messenger RNA expression (P < 0.05) and downstream NF-κB signaling compared with PLA. Conclusion Protein-polyphenol drink ingestion likely accelerates recovery of muscle function by attenuating inflammatory NF-κB transcriptional signaling, possibly to reduce aberrant tissue degradation rather than increase myoPS rates.

Background Pre-exercise supplements containing low doses of caffeine improve endurance exercise performance, but the most efficacious time for consumption before intense endurance exercise remains unclear, as does the contribution of caffeine metabolism. Methods This study assessed the timing of a commercially available supplement containing 200 mg of caffeine, 1600 mg of β-alanine and 1000 mg of quercetin [Beachbody Performance Energize, Beachbody LLC, USA] on exercise performance, perception of effort and plasma caffeine metabolites. Thirteen cyclists (V̇O2max 64.5 ± 1.4 ml kg− 1 min− 1 (± SEM)) completed four experimental visits consisting of 30 min of steady-state exercise on a cycle ergometer at 83 ± 1% V̇O2max followed by a 15-min time trial, with perceived exertion measured regularly. On three of the visits, participants consumed caffeine either 35 min before steady-state exercise (PRE), at the onset of steady-state (ONS) or immediately before the time trial (DUR) phases, with a placebo consumed at the other two time points (i.e. three drinks per visit). The other visit (PLA) consisted of consuming the placebo supplement at all three time points. The placebo was taste-, colour- and calorie-matched. Results Total work performed during the time trial in PRE was 5% greater than PLA (3.53 ± 0.14 vs. 3.36 ± 0.13 kJ kg− 1 body mass; P = 0.0025), but not ONS (3.44 ± 0.13 kJ kg− 1; P = 0.3619) or DUR (3.39 ± 0.13 kJ kg− 1; P = 0.925), which were similar to PLA. Perceived exertion was lowest during steady-state exercise in the PRE condition (P P Conclusion In summary, ingestion of a pre-exercise supplement containing 200 mg caffeine 35 min before exercise appeared optimal for improved performance in a subsequent fatiguing time trial, possibly by reducing the perception of effort. Whether this was due to increased circulating paraxanthine requires further investigation. Trial registration ClinicalTrials.Gov,NCT02985606; 10/26/2016.

OBJECTIVES: The ability of a novel beadlet-in-oil, gastric-resistant, vegetarian capsule containing a multiple vitamin-mineral (MVM) composition to deliver capsule contents past the stomach was tested by standard in vitro tablet disintegration procedures using magnesium as the marker of capsule contents dissolution. METHODS: A novel capsule design using a gastric-resistant vegetarian hypromellose/gellan gum capsule (DRcaps®, Capsugel®) was tested for disintegration in a standard tablet disintegration apparatus according to compendial United States Pharmacopeia methods, as per Good Manufacturing Practices for dietary supplements. The MVM ingredients were encapsulated into size 0 Vcaps® (hypromellose with no gastric acid resistance) and DRcaps®. Individual capsules were placed into chambers containing simulated gastric fluid (0.1 M HCl) for 120 minutes then changed to simulated intestinal fluid (buffered 2% sodium lauryl sulfate, pH 6.0) for an additional 300 minutes. Aliquots were tested for magnesium concentration at ten time points by atomic absorption spectrometry. RESULTS: Magnesium was contained inside coated beadlets along with ferrous bisglycinate, methylcobalamin, 5-methyltetrahydrofolate, calcium fructoborate and cellulose. Vcaps® released 50% of the magnesium between 30–45 minutes and all by 60 minutes in the acid phase. DRcaps® released 25% of the magnesium at 45 minutes, and 43% at 120 minutes, followed by slow, steady release of the remaining magnesium by 420 minutes. CONCLUSIONS: These dissolution profiles reproduce the known, rapid disintegration profile of Vcaps® when wetted. DRcaps® released the majority of their contents after the pH was changed to intestinal conditions, and then the beadlets released the water-soluble ingredients (magnesium) in a linear manner over a two hour period. Since normal stomach emptying of DRcaps® without a meal is less than 20–30 minutes (previously shown), DRcap® MVMs bypass the stomach almost completely to release ingredients in the small intestine. Thus, a novel, beadlet-in-oil, gastric-resistant capsule delivered its contents past the stomach. These properties have the ability to improve tolerability and thus, compliance with users. FUNDING SOURCES: Capsugel®, Greenwood, SC, conducted this study for Ritual. SUPPORTING TABLES, IMAGES AND/OR GRAPHS

OBJECTIVES: The dietary supplement industry has grown substantially, and advances in technology have played important roles in the development of new forms of dietary supplements and delivery systems. While it is recommended to use compendia methods for testing dietary supplements, the analytical methods in place are often developed for single ingredients or simple matrices and are not always fit to be used for solving modern matrix challenges. Oil- and beadlet-filled, gastric acid-resistant capsule (OBGRC) is designed to improve nutrient delivery and absorption. However, it is challenging to accurately analyze labile nutrients in OBGRCs because nutrients within different physical locations are difficult to separate, and lengthy sample preparations lead to nutrient degradation. We developed and validated analytical methods to determine methylcobalamin (MeB12), 5-methyltetrahydrofolate (5-MTHF), and trans menaquinone-7 (MK-7) in OBGRCs. METHODS: MeB12 and 5-MTHF were extracted by completely dissolving the OBGRC shells using aqueous buffers and then applying hexane-facilitated liquid-liquid extraction to remove the oil phase and determined by RP-HPLC using superficially porous particles columns. For MK-7 analysis, the whole OBGRC was cut to open and the oil phase was collected to dissolve in a mixture of dimethyl sulfoxide/tetrahydrofuran/ethanol before analyzed by RP-HPLC with fluorescence detection after on-line, post-column zinc reduction. The methods were subjected to single-laboratory validations according to the United States Pharmacopeia (USP) guidelines for linearity, suitability, detection limits, specificity, accuracy, and precision. RESULTS: The methods achieved chromatographic resolution of target analytes without the expensive requirement of ultra-high pressure liquid chromatography and/or mass spectrometry. The methods have wide analytical ranges (from at least 50% to 250% of the input concentrations), high precision (repeatability relative standard deviations ranging from 1.04% to 4.90%), and high accuracy (spike recovery rate ranging from 89.2% to 108%). CONCLUSIONS: All three methods passed USP method validation criteria and have the potential to be widely adopted to analyze MeB12, 5-MTHF, and MK-7 in similar matrices for quality control purposes. FUNDING SOURCES: The research funding was provided by Ritual and Eurofins.

OBJECTIVES: We determined if multivitamin supplementation (MV) affected serum folate levels in women during a 12-week intervention, and if genotypes affected these outcomes. METHODS: Women (21–40 years old) were recruited from the local community. PRE testing occurred between 0600–0900 AM 5 ± 2 days following each participants’ last menstrual cycle, and involved a buccal cell collection and a fasted blood draw. Participants were then randomly assigned in a double-blinded fashion to the MV group (n = 43) or placebo group (n = 51). MV (per 2 beadlets-in-capsule serving) contained 50 mcg vitamin D3, 6.7 mg alpha tocopherol of vitamin E as mixed tocopherols, 90 μg vitamin K2 MK7, 600 μg 5-methyltetrahydrofolate, 8 μg B12, 8 mg iron, 50 mg magnesium, 1 mg boron, and 320 mg omega-3 fatty acids. The placebo capsules contained safflower oil and cellulose. Participants consumed two capsules per day with breakfast for 12 weeks. Following the intervention, participants reported to the laboratory for POST testing. Serum folate analyses were performed at a hematology laboratory (East Alabama Medical Center, Opelika, AL, USA). SNPs related to folate metabolism (MTHFR rs1801133, MTHFR rs1801131, MTHFR rs2274976, MTRR rs1801394) were interrogated using the Axiom PMDA.r6 array (Thermo Fisher Scientific, Waltham, MA, USA). RESULTS: A group × time interaction was observed for serum folate levels and dietary folate intake (P

Background The glycemic index (GI) is used to classify carbohydrate-containing foods and products according to their effect on postprandial blood glucose responses. Low GI products release carbohyd...

Objective The mechanistic role of the ubiquitin ligases atrogin-1 and MuRF1 in glucocorticoid-induced muscle wasting is not fully understood. Here, we tested the hypothesis that glucocorticoid-induced muscle atrophy is at least in part linked to atrogin-1 and MuRF1 expression and that the ubiquitin ligases are regulated by compensatory mechanisms. Methods The expression of atrogin-1 and MuRF1 was suppressed individually or in combination in cultured L6 myotubes by using siRNA technique. Myotubes were treated with dexamethasone followed by determination of mRNA and protein levels for atrogin-1 and MuRF1, protein synthesis and degradation rates, and myotube morphology. Results Suppression of atrogin-1 resulted in increased expression of MuRF1 and vice versa, suggesting that the ubiquitin ligases are regulated by compensatory mechanisms. Simultaneous suppression of atrogin-1 and MuRF1 resulted in myotube hypertrophy, mainly reflecting stimulated protein synthesis, and prevented dexamethasone-induced myotube atrophy, mainly reflecting inhibited protein degradation. Conclusions The results provide evidence for a link between upregulated atrogin-1 and MuRF1 expression and glucocorticoid-induced muscle atrophy. The study also suggests that atrogin-1 and MuRF1 levels are regulated by compensatory mechanisms and that inhibition of both ubiquitin ligases may be needed to prevent glucocorticoid-induced muscle proteolysis and atrophy.

We review recent evidence that acetylation and deacetylation of cellular proteins, including transcription factors and nuclear cofactors, may be involved in the regulation of muscle mass. The level of protein acetylation is balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs) and studies suggest that this balance is perturbed in muscle wasting. Hyperacetylation of transcription factors and nuclear cofactors regulating gene transcription in muscle wasting may influence muscle mass. In addition, hyperacetylation may render proteins susceptible to degradation by different mechanisms, including intrinsic ubiquitin ligase activity exerted by HATs and by dissociation of proteins from cellular chaperones. In recent studies, inhibition of p300/HAT expression and activity and stimulation of SIRT1-dependent HDAC activity reduced glucocorticoid-induced catabolic response in skeletal muscle, providing further evidence that hyperacetylation plays a role in muscle wasting. It should be noted, however, that although several studies advocate a role of hyperacetylation in muscle wasting, apparently contradictory results have also been reported. For example, muscle atrophy caused by denervation or immobilization may be associated with reduced, rather than increased, protein acetylation. In addition, whereas hyperacetylation results in increased degradation of certain proteins, other proteins may be stabilized by increased acetylation. Thus, the role of acetylation and deacetylation in the regulation of muscle mass may be both condition- and protein-specific. The influence of HATs and HDACs on the regulation of muscle mass, as well as methods to modulate protein acetylation, is an important area for continued research aimed at preventing and treating muscle wasting.

Resveratrol (3,5,4’-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.

Muscle wasting in patients with sepsis, severe injury, and cancer is associated with increased transcription of several genes regulating different proteolytic pathways. The involvement of gene activation in muscle wasting suggests that transcription factors and nuclear cofactors play important roles in the regulation of muscle mass. Among transcription factors, NF-κB, C/EBPβ, and FOXO transcription factors are activated in atrophying muscle and stimulate the transcription of genes in the ubiquitin-proteasome proteolytic pathway, as well as genes regulating authophagy/lysosomal proteolysis. Changes in the expression and activity of several nuclear cofactors, including the histone acetyltransferase p300, histone deacetylases (HDACs), such as HDAC3, HDAC6, and SIRT1, as well as the nuclear cofactors PGC-1α and β, contribute to loss of muscle mass in various catabolic conditions. The activity of transcription factors and nuclear cofactors involved in the regulation of muscle mass is influenced not only by their abundance, but also by posttranslational modifications as well, including ubiquitination, phosphorylation, and acetylation. Transcription factors and nuclear cofactors involved in muscle wasting interact with each other at multiple levels, supporting the concept that the molecular regulation of muscle mass in various catabolic conditions is complex. An increased understanding of molecules that modulate gene transcription in catabolic muscle may make it possible to develop treatments targeting transcription factors and nuclear cofactors in the prevention and treatment of muscle wasting.

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis-induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham-operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p-Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p-Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p-Smad2 levels. Of note, total and p-Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well.

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 but it is not known if atrogin-1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin-1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain-, and caspase-3-dependent protein breakdown in addition to proteasome-dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin-1 and MuRF1 mRNA levels. The same treatment increased proteasome-, cathepsin L-, and calpain-dependent proteolytic rates by approximately 40% but did not influence caspase-3-dependent proteolysis. The expression of atrogin-1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley-Liss, Inc.

Sepsis, severe injury, and cancer are associated with loss of muscle mass. Muscle wasting in these conditions is mainly caused by increased proteolysis, at least in part regulated by nuclear factor-kappaB. Despite recent progress in the understanding of mediators and mechanisms involved in muscle wasting, effective and universally accepted treatments by which muscle atrophy can be prevented or reversed are still lacking. We review recent evidence suggesting that curcumin (diferuloylmethane), a component of the spice turmeric, may prevent loss of muscle mass during sepsis and endotoxemia and may stimulate muscle regeneration after traumatic injury. Curcumin has been part of the traditional Asian medicine for centuries, mainly because of its anti-inflammatory properties. Studies suggest that inhibition of nuclear factor-kappaB is one of the mechanisms by which curcumin exerts its ant-inflammatory effects. Curcumin is easily accessible, inexpensive, and non-toxic even at high doses, and may therefore offer an important treatment modality in muscle wasting and injury. It should be noted, however, that the muscle-sparing effects of curcumin are not universally accepted, and more studies are therefore needed to further test the role of curcumin in the prevention and treatment of muscle wasting.

A characteristic manifestation of sepsis is muscle lactate accumulation. This study examined any putative (causative) association between pyruvate dehydrogenase complex (PDC) inhibition and lactate accumulation in the extensor digitorum longus (EDL) muscle of rats infused with lipopolysaccharide (LPS), and explored the involvement of increased transcription of muscle-specific pyruvate dehydrogenase kinase (PDK) isoenzymes. Conscious, male Sprague-Dawley rats were infused i.v. with saline (0.4 ml h(-1), control) or LPS (150 mug kg(-1) h(-1)) for 2 h, 6 h or 24 h (n = 6-8). Muscle lactate concentration was elevated after 2, 6 and 24 h LPS infusion. Muscle PDC activity was the same at 2 h and 6 h, but was 65% lower after 24 h of LPS infusion (P < 0.01), when there was a 47% decrease in acetylcarnitine concentration (P < 0.05), and a 24-fold increase in PDK4 mRNA expression (P < 0.001). These changes were preceded by marked increases in tumour necrosis factor-alpha and interleukin-6 mRNA expression at 2 h. The findings indicate that the early (2 and 6 h) elevation in muscle lactate concentration during LPS infusion was not attributable to limited muscle oxygen availability or ATP production (evidenced by unchanged ATP and phosphocreatine (PCr) concentrations) or to PDC inhibition, whereas after 24 h, muscle lactate accumulation appears to have resulted from PDC activation status limiting pyruvate flux, most probably due to cytokine-mediated up-regulation of PDK4 transcription.

FOXO1 is involved in glucocorticoid- and sepsis-induced muscle wasting, in part reflecting regulation of atrogin-1 and MuRF1. Mechanisms influencing FOXO1 expression in muscle wasting are poorly understood. We hypothesized that the transcription factor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) upregulates muscle FOXO1 expression and activity with a downstream upregulation of atrogin-1 and MuRF1 expression during sepsis and glucocorticoid treatment and that inhibition of PPARβ/δ activity can prevent muscle wasting. We found that activation of PPARβ/δ in cultured myotubes increased FOXO1 activity, atrogin-1 and MuRF1 expression, protein degradation and myotube atrophy. Treatment of myotubes with dexamethasone increased PPARβ/δ expression and activity. Dexamethasone-induced FOXO1 activation and atrogin-1 and MuRF1 expression, protein degradation, and myotube atrophy were inhibited by PPARβ/δ blocker or siRNA. Importantly, muscle wasting induced in rats by dexamethasone or sepsis was prevented by treatment with a PPARβ/δ inhibitor. The present results suggest that PPARβ/δ regulates FOXO1 activation in glucocorticoid- and sepsis-induced muscle wasting and that treatment with a PPARβ/δ inhibitor may ameliorate loss of muscle mass in these conditions.

Sepsis is associated with impaired muscle function but the role of glucocorticoids in sepsis-induced muscle weakness is not known. We tested the role of glucocorticoids in sepsis-induced muscle weakness by treating septic rats with the glucocorticoid receptor antagonist RU38486. In addition, normal rats were treated with dexamethasone to further examine the role of glucocorticoids in the regulation of muscle strength. Sepsis was induced in rats by cecal ligation and puncture, and muscle force generation (peak twitch and tetanic tension) was determined in lower extremity muscles. In other experiments, absolute and specific force as well as stiffness (reflecting the function of actomyosin cross bridges) were determined in isolated skinned muscle fibers from control and septic rats. Sepsis and treatment with dexamethasone resulted in reduced maximal twitch and tetanic force in intact isolated extensor digitorum longus muscles. The absolute and specific maximal force in isolated muscle fibers was reduced during sepsis together with decreased fiber stiffness. These effects of sepsis were blunted (but not abolished) by RU38486. The results suggest that muscle weakness during sepsis is at least in part regulated by glucocorticoids and reflects loss of contractility at the cellular (individual muscle fiber) level. In addition, the results suggest that reduced function of the cross bridges between actin and myosin (documented as reduced muscle fiber stiffness) may be involved in sepsis-induced muscle weakness. An increased understanding of mechanisms involved in loss of muscle strength will be important for the development of new treatment strategies in patients with this debilitating consequence of sepsis.

Recent studies suggest that the expression and activity of the histone acetyltransferase p300 are upregulated in catabolic muscle allowing for acetylation of cellular proteins. The function of transcription factors is influenced by posttranslational modifications, including acetylation. It is not known if transcription factors involved in the regulation of muscle mass are acetylated in atrophying muscle. We determined cellular levels of acetylated C/EBPβ, C/EBPδ, FOXO1, FOXO3a, and NF-kB/p65 in dexamethasone-treated L6 muscle cells, a commonly used in vitro model of muscle wasting. The role of p300 in dexamethasone-induced transcription factor acetylation and myotube atrophy was examined by transfecting muscle cells with p300 siRNA. Treatment of L6 myotubes with dexamethasone resulted in increased cellular levels of acetylated C/EBPβ and δ, FOXO1 and 3a, and p65. Downregulation of p300 with p300 siRNA reduced acetylation of transcription factors and decreased dexamethasone-induced myotube atrophy and expression of the ubiquitin ligase MuRF1. The results suggest that several muscle wasting-related transcription factors are acetylated supporting the concept that posttranslational modifications of proteins regulating gene transcription may be involved in the loss of muscle mass. The results also suggest that acetylation of the transcription factors is at least in part regulated by p300 and plays a role in glucocorticoid-induced muscle atrophy. Targeting molecules that regulate acetylation of transcription factors may help reduce the impact of muscle wasting.

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.

Muscle wasting in catabolic patients is in part mediated by glucocorticoids and is associated with increased expression and activity of the transcription factor C/EBPβ. It is not known, however, if C/EBPβ is causally linked to glucocorticoid-induced muscle atrophy. We used dexamethasone-treated L6 myoblasts and myotubes to test the role of C/EBPβ in glucocorticoid-induced expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1, protein degradation, and muscle atrophy by transfecting cells with C/EBPβ siRNA. In myoblasts, silencing C/EBPβ expression with siRNA inhibited dexamethasone-induced increase in protein degradation, atrogin-1 and MuRF1 expression, and muscle cell atrophy. Similar effects of C/EBPβ siRNA were seen in myotubes except that the dexamethasone-induced increase in MuRF1 expression was not affected by C/EBPβ siRNA in myotubes. In additional experiments, overexpressing C/EBPβ did not influence atrogin-1 or MuRF1 expression in myoblasts or myotubes. Taken together, our observations suggest that glucocorticoid-induced muscle wasting is at least in part regulated by C/EBPβ. Increased C/EBPβ expression alone, however, is not sufficient to upregulate atrogin-1 and MuRF1 expression.

Muscle wasting during sepsis is at least in part regulated by glucocorticoids and is associated with increased transcription of genes encoding the ubiquitin ligases atrogin-1 and muscle-specific RING-finger protein-1 (MuRF1). Recent studies suggest that muscle atrophy caused by denervation is associated with reduced expression of the nuclear cofactor peroxisome proliferator-activated receptor-γ coactivator (PGC)-1β and that PGC-1β may be a repressor of the atrogin-1 and MuRF1 genes. The influence of other muscle-wasting conditions on the expression of PGC-1β is not known. We tested the influence of sepsis and glucocorticoids on PGC-1β and examined the potential link between downregulated PGC-1β expression and upregulated atrogin-1 and MuRF1 expression in skeletal muscle. Sepsis in rats and mice and treatment with dexamethasone resulted in downregulated expression of PGC-1β and increased expression of atrogin-1 and MuRF1 in the fast-twitch extensor digitorum longus muscle, with less pronounced changes in the slow-twitch soleus muscle. In additional experiments, adenoviral gene transfer of PGC-1β into cultured C2C12 myotubes resulted in a dose-dependent decrease in atrogin-1 and MuRF1 mRNA levels. Treatment of cultured C2C12 myotubes with dexamethasone or PGC-1β small interfering RNA (siRNA) resulted in downregulated PGC-1β expression and increased protein degradation. Taken together, our results suggest that sepsis- and glucocorticoid-induced muscle wasting may, at least in part, be regulated by decreased expression of the nuclear cofactor PGC-1β.

Muscle wasting during sepsis is in part regulated by glucocorticoids. In recent studies, treatment of cultured muscle cells in vitro with dexamethasone upregulated expression and activity of p300, a histone acetyl transferase (HAT), and reduced expression and activity of the histone deacetylases-3 (HDAC3) and -6, changes that favor hyperacetylation. Here, we tested the hypothesis that sepsis and glucocorticoids regulate p300 and HDAC3 and -6 in skeletal muscle in vivo. Because sepsis-induced metabolic changes are particularly pronounced in white, fast-twitch skeletal muscle, most experiments were performed in extensor digitorum longus muscles. Sepsis in rats upregulated p300 mRNA and protein levels, stimulated HAT activity, and reduced HDAC6 expression and HDAC activity. The sepsis-induced changes in p300 and HDAC expression were prevented by the glucocorticoid receptor antagonist RU38486. Treatment of rats with dexamethasone increased expression of p300 and HAT activity, reduced expression of HDAC3 and -6, and inhibited HDAC activity. Finally, treatment with the HDAC inhibitor trichostatin A resulted in increased muscle proteolysis and expression of the ubiquitin ligase atrogin-1. Taken together, our results suggest for the first time that sepsis-induced muscle wasting may be regulated by glucocorticoid-dependent hyperacetylation caused by increased p300 and reduced HDAC expression and activity. The recent development of pharmacological HDAC activators may provide a novel avenue to prevent and treat muscle wasting in sepsis and other catabolic conditions.

The purpose of this review is to discuss novel insight into mechanisms of glucocorticoid-regulated muscle wasting, in particular the role of transcription factors and nuclear cofactors. In addition, novel strategies that may become useful in the treatment or prevention of glucocorticoid-induced muscle wasting are reviewed.Studies suggest that glucocorticoid-induced upregulation of the transcription factors Forkhead box O 1 and CCAAT/enhancer-binding protein beta and downregulation of MyoD and myogenin are involved in glucocorticoid-induced muscle wasting. In addition, glucocorticoid-induced hyperacetylation caused by increased expression of the nuclear cofactor p300 and its histone acetyl transferase activity and decreased expression and activity of histone deacetylases plays an important role in glucocorticoid-induced muscle proteolysis and wasting. Other mechanisms may also be involved in glucocorticoid-induced muscle wasting, including insulin resistance and store-operated calcium entry. Novel potential strategies to prevent or treat glucocorticoid-induced muscle wasting include the use of small molecule histone deacetylase activators, dissociated glucocorticoid receptor agonists, and 11beta-hydroxysteroid dehydrogenase type 1 inhibitors.An increased understanding of molecular mechanisms regulating glucocorticoid-induced muscle wasting will help develop new strategies to prevent and treat this debilitating condition.

Sepsis-induced muscle wasting has severe clinical consequences, including muscle weakness, need for prolonged ventilatory support and stay in the intensive care unit, and delayed ambulation with risk for pulmonary and thromboembolic complications. Understanding molecular mechanisms regulating loss of muscle mass in septic patients therefore has significant clinical implications. Forkhead Box O (FOXO) transcription factors have been implicated in muscle wasting, partly reflecting upregulation of the ubiquitin ligases atrogin-1 and MuRF1. The influence of sepsis on FOXO transcription factors in skeletal muscle is poorly understood. We tested the hypothesis that sepsis upregulates expression and activity of FOXO transcription factors in skeletal muscle by a glucocorticoid-dependent mechanism. Sepsis in rats increased muscle FOXO1 and 3a mRNA and protein levels but did not influence FOXO4 expression. Nuclear FOXO1 levels and DNA binding activity were increased in septic muscle whereas FOXO3a nuclear levels were not increased during sepsis. Sepsis-induced expression of FOXO1 was reduced by the glucocorticoid receptor antagonist RU38486 and treatment of rats with dexamethasone increased FOXO1 mRNA levels suggesting that the expression of FOXO1 is regulated by glucocorticoids. Reducing FOXO1, but not FOXO3a, expression by siRNA in cultured L6 myotubes inhibited dexamethasone-induced atrogin-1 and MuRF1 expression, further supporting a role of FOXO1 in glucocorticoid-regulated muscle wasting. Results suggest that sepsis increases FOXO1 expression and activity in skeletal muscle by a glucocorticoid-dependent mechanism and that glucocorticoid-dependent upregulation of atrogin-1 and MuRF1 in skeletal muscle is regulated by FOXO1. The study is significant because it provides novel information about molecular mechanisms involved in sepsis-induced muscle wasting.

We tested the hypothesis that treatment of rats with curcumin prevents sepsis-induced muscle protein degradation. In addition, we determined the influence of curcumin on different proteolytic pathways that are activated in septic muscle (i.e., ubiquitin-proteasome-, calpain-, and cathepsin L-dependent proteolysis) and examined the role of NF-κB and p38/MAP kinase inactivation in curcumin-induced inhibition of muscle protein breakdown. Rats were made septic by cecal ligation and puncture or were sham-operated. Groups of rats were treated with three intraperitoneal doses (600 mg/kg) of curcumin or corresponding volumes of solvent. Protein breakdown rates were measured as release of tyrosine from incubated extensor digitorum longus muscles. Treatment with curcumin prevented sepsis-induced increase in muscle protein breakdown. Surprisingly, the upregulated expression of the ubiquitin ligases atrogin-1 and MuRF1 was not influenced by curcumin. When muscles from septic rats were treated with curcumin in vitro, proteasome-, calpain-, and cathepsin L-dependent protein breakdown rates were reduced, and nuclear NF-κB/p65 expression and activity as well as levels of phosphorylated (activated) p38 were decreased. Results suggest that sepsis-induced muscle proteolysis can be blocked by curcumin and that this effect may, at least in part, be caused by inhibited NF-κB and p38 activities. The results also suggest that there is not an absolute correlation between changes in muscle protein breakdown rates and changes in atrogin-1 and MuRF1 expression during treatment of muscle wasting.

BACKGROUND:It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia. METHODOLOGY/PRINCIPAL FINDINGS:To investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 microg kg(-1) h(-1)) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P