Skeletal Muscle Protein Turnover Research Articles

Heterochronic Plasma Transfer Alters Proteostatic Maintenance in Skeletal Muscle

Studies using heterochronic blood transfer (HBT) and heterochronic plasma transfer (HPT) show that circulating factors in young blood and plasma can rejuvenate the ability of aged skeletal muscle to respond to damage. Additionally, the transfer of old blood and plasma to young mice inhibits skeletal muscle regeneration. These changes in the ability of skeletal muscle to regenerate in response to modifications in the systemic environment are largely mediated by satellite cells. However, the role of satellite cells in maintaining normal muscle function is unclear. Therefore, how the systemic environment influences skeletal muscle in the absence of extreme muscle damage is still unknown. Proteostatic maintenance, which includes protein synthesis and breakdown, is necessary for cellular stability in skeletal muscle. One measure of proteostatic maintenance is protein:DNA ratio (i.e. how much protein is synthesized for cellular maintenance verses cellular proliferation). Here we determined if altering the systemic environment of old mice with young plasma and the systemic environment of young mice with old plasma would change proteostatic maintenance of skeletal muscle. We hypothesized that compared to old mice that receive old plasma (OOp), old mice that underwent HPT from young mice (OYp) would improve proteostatic maintenance. Additionally, we hypothesized that compared to young mice that receive young plasma (YYp), young mice that underwent HPT from old mice (YOp) would decrease proteostatic maintenance. To test this, we implanted young (5-month, n=3 YYp and 5 YOp) and old (24-months, n=5 OOp, and 5 OYp) C57BL/6J mice with a jugular catheter. All mice underwent heterochronic plasma transfer (HPT), receiving 100μl plasma injections, every three days for 24 days. The day before HPT, mice began stable isotope labeling with a bolus injection of 99% deuterium oxide (D2O) followed by 8% D2O-enriched drinking water. After 4 weeks, the quadriceps, gastrocnemius, and tibialis anterior muscles were harvested and analyzed for DNA and protein synthesis to calculate the protein:DNA ratios. The protein:DNA ratio for the mitochondrial fraction trended toward a higher ratio in the tibialis anterior of the OYp versus OOp (11.93 ± 1.161 and 8.928 ± 1.128, respectively: p=0.10). The protein:DNA ratio for the myofibrillar fraction trended toward being lower in the gastrocnemius of the YOp compared to YYp (8.338 ± 0.429 and 6.281 ± 0.737, respectively: p=0.06). These results suggest that manipulating the systemic environment with young or old plasma can change proteostatic maintenance of skeletal muscle so that the muscle reflects the systemic environment. Therefore, strategies to improve the systemic environment, like with exercise, may be a feasible strategy to improve proteostatic maintenance in aged skeletal muscle. Further research is needed to determine what circulating factors alter protein and DNA turnover in skeletal muscle.

PHD3 mediates denervation skeletal muscle atrophy through Nf-κB signal pathway.

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P<.05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P<.05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P<.05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P<.05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P<.05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P<.05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P<.05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

JNK signaling contributes to skeletal muscle wasting and protein turnover in pancreatic cancer cachexia.

Cancer cachexia patients experience significant muscle wasting, which impairs the quality of life and treatment efficacy for patients. Skeletal muscle protein turnover is imparted by increased expression of ubiquitin-proteasome pathway components. Mitogen-activated protein kinases p38 and ERK have been shown to augment E3 ubiquitin ligase expression. Utilizing reverse-phase protein arrays, we identified pancreatic cancer cell-conditioned media-induced activation of JNK signaling in myotubes differentiated from C2C12 myoblasts. Inhibition of JNK signaling with SP600125 reduced cancer cell-conditioned media-induced myotube atrophy, myosin heavy chain protein turnover, and mRNA expression of cachexia-specific ubiquitin ligases Trim63 and Fbxo32. Furthermore, utilizing an orthotopic pancreatic cancer cachexia mouse model, we demonstrated that treatment of tumor-bearing mice with SP600125 improved longitudinal measurements of forelimb grip strength. Post-necropsy measurements demonstrated that SP600125 treatment rescued body weight, carcass weight, and gastrocnemius muscle weight loss without impacting tumor growth. JNK inhibitor treatment also rescued myofiber degeneration and reduced the muscle expression of Trim63 and Fbxo32. These data demonstrate that JNK signaling contributes to muscle wasting in cancer cachexia, and its inhibition has the potential to be utilized as an anti-cachectic therapy.

Serum extracellular vesicle miR-203a-3p content is associated with skeletal muscle mass and protein turnover during disuse atrophy and regrowth.

Small noncoding microRNAs (miRNAs) are important regulators of skeletal muscle size, and circulating miRNAs within extracellular vesicles (EVs) may contribute to atrophy and its associated systemic effects. The purpose of this study was to understand how muscle atrophy and regrowth alter in vivo serum EV miRNA content. We also associated changes in serum EV miRNA with protein synthesis, protein degradation, and miRNA within muscle, kidney, and liver. We subjected adult (10 mo) F344/BN rats to three conditions: weight bearing (WB), hindlimb suspension (HS) for 7 days to induce muscle atrophy, and HS for 7 days followed by 7 days of reloading (HSR). Microarray analysis of EV miRNA content showed that the overall changes in serum EV miRNA were predicted to target major anabolic, catabolic, and mechanosensitive pathways. MiR-203a-3p was the only miRNA demonstrating substantial differences in HS EVs compared with WB. There was a limited association of EV miRNA content to the corresponding miRNA content within the muscle, kidney, or liver. Stepwise linear regression demonstrated that EV miR-203a-3p was correlated with muscle mass and muscle protein synthesis and degradation across all conditions. Finally, EV miR-203a-3p expression was significantly decreased in human subjects who underwent unilateral lower limb suspension (ULLS) to induce muscle atrophy. Altogether, we show that serum EV miR-203a-3p expression is related to skeletal muscle protein turnover and atrophy. We suggest that serum EV miR-203a-3p content may be a useful biomarker and future work should investigate whether serum EV miR-203a-3p content is mechanistically linked to protein synthesis and degradation.

Skeletal Muscle Raptor And Tsc1/2 Differential Responses To Activity And Fasting In The Tumor Environment

Cancer is a debilitating disease that is often accompanied by decreased physical activity and chronic energetic stress that disrupts muscle proteostasis. Skeletal muscle protein turnover is highly sensitive to changes in feeding and activity. Raptor serine 792 and TSC1/2 serine 1387 phosphorylation sites, when activated, can inhibit mTORC1 activation leading to suppressed anabolic signaling, and has been implicated in the regulation of skeletal muscle wasting with cancer. PURPOSE: To examine the effect of a 12-hour fast on the phosphorylation of Raptor and TSC1/2 in male ApcMin/+ mice, and if voluntary wheel activity can alter the fasting response. METHODS: Male C57BL/6 (B6, N=24) and ApcMin/+ (MIN, N=31) mice were either sacrificed under ad libitum conditions (B6-fed, M-fed), fasted for 12hrs (B6-fast, M-fast), or fasted for 12hrs following 4wks of voluntary wheel running (B6+W, M+W). TSC1/2 serine 1387 and Raptor serine 792 were measured in the gastrocnemius muscle as phosphorylation to total ratio by western blot. Protein synthesis was measured by puromycin incorporation. RESULTS: All MIN mice exhibited body weight loss (p<0.001) and reduced gastrocnemius mass (p<0.001) when compared to all B6 mice. Raptor phosphorylation (pRaptor) was induced in M-fast compared to M-fed (p=0.019), but there was no change in B6+fast compared to B6-fed (p=0.414). TSC1/2 phosphorylation was induced in M-fast compared to M-fed (p=0.001) and in B6-fast to B6-fed (p=0.039). Puromycin was trending to be reduced in M-fast compared to MIN-fed (p=0.070), but there was no change in B6-fast to B6-fed (p=0.323). Raptor phosphorylation was not different in M-fast compared to M+W (p=0.302), however pRaptor was reduced in B6+W compared B6-fast (p=0.028). TSC1/2 phosphorylation (p=0.001) and puromycin (p=0.016) were induced in MIN+W compared to M-fast, with no changes in the B6-fast to B6+W (p=0.360, p=0.196; respectively). CONCLUSIONS: Our results provide evidence that the cancer environment disrupts anabolic suppression of mTORC1 in skeletal muscle. Interestingly, TSC1/2 phosphorylation was sensitive to fasting independent of the tumor environment. Wheel activity induced protein synthesis independent of Raptor phosphorylation therefore, further studies are warranted to determine this specific mechanism.

Blood flow-restricted resistance exercise alters the surface profile, miRNA cargo and functional impact of circulating extracellular vesicles

Ischemic exercise conducted as low-load blood flow restricted resistance exercise (BFRE) can lead to muscle remodelling and promote muscle growth, possibly through activation of muscle precursor cells. Cell activation can be triggered by blood borne extracellular vesicles (EVs) as these nano-sized particles are involved in long distance signalling. In this study, EVs isolated from plasma of healthy human subjects performing a single bout of BFRE were investigated for their change in EV surface profiles and miRNA cargos as well as their impact on skeletal muscle precursor cell proliferation. We found that after BFRE, five EV surface markers and 12 miRNAs were significantly altered. Furthermore, target prediction and functional enrichment analysis of the miRNAs revealed several target genes that are associated to biological pathways involved in skeletal muscle protein turnover. Interestingly, EVs from BFRE plasma increased the proliferation of muscle precursor cells. In addition, alterations in surface markers and miRNAs indicated that the combination of exercise and ischemic conditioning during BFRE can stimulate blood cells to release EVs. These results support that BFRE promotes EV release to engage in muscle remodelling and/or growth processes.

Exercise Improves the Fasting Regulation of Skeletal Muscle Protein Synthesis in Tumor Bearing Mice

IntroductionCancer is a debilitating disease that is often accompanied by decreased physical activity and chronic energetic stress that disrupts muscle proteostasis. Skeletal muscle protein turnover is highly sensitive to changes in feeding and activity. Raptor (Ser 792) and TSC1/2 (Ser 1387) phosphorylation can inhibit mTORC1 activation and suppress anabolic signaling, which has been implicated in cancer‐induced skeletal muscle wasting.PurposeTo examine the effect of the cancer environment on the fasting regulation of Raptor and TSC1/2 in male ApcMin/+ mice and determine if increased physical activity could alter this response.MethodsMale C57BL/6 (B6, N=24) and ApcMin/+ (MIN, N=31) mice were either sacrificed under ad libitum conditions (B6‐fed, M‐fed), fasted for 12hrs (B6‐fast, M‐fast), or fasted for 12hrs following 4wks of voluntary wheel running (B6+W, M+W). TSC1/2 serine 1387 and Raptor serine 792 were measured in the gastrocnemius muscle as phosphorylation to total ratio by western blot. Protein synthesis was measured by puromycin incorporation.ResultsAll MIN mice exhibited body weight loss (p&lt;0.001) and reduced gastrocnemius mass (p&lt;0.001) when compared to all B6 mice. Raptor phosphorylation (pRaptor) was induced in M‐fast compared to M‐fed (p=0.019), which was not changed by fasting in B6 mice (p=0.414). TSC1/2 phosphorylation was induced in M‐fast compared to M‐fed (p=0.001) and in B6‐fast to B6‐fed (p=0.039). Puromycin was trending to be reduced in M‐fast compared to MIN‐fed (p=0.070), but there was no change in B6‐fast to B6‐fed (p=0.323). Raptor phosphorylation was not different in M‐fast compared to M+W (p=0.302), however pRaptor was reduced in B6+W compared B6‐fast (p=0.028). TSC1/2 phosphorylation (p=0.001) and puromycin (p=0.016) were induced in MIN+W compared to M‐fast, with no changes in the B6‐fast to B6+W (p=0.360, p=0.196; respectively).ConclusionsOur results provide evidence that the cancer environment disrupts fasting regulation of mTORC1 in skeletal muscle. Interestingly, TSC1/2 phosphorylation was sensitive to fasting independent of the tumor environment, while wheel activity induced protein synthesis independent of Raptor phosphorylation. Therefore, further study is warranted to determine physical activities regulation of fasting skeletal muscle protein synthesis in tumor bearing mice.Support or Funding InformationAcknowledgementsNCI R01‐CA121249

Massage as a mechanotherapy promotes skeletal muscle protein and ribosomal turnover but does not mitigate muscle atrophy during disuse in adult rats.

Interventions that decrease atrophy during disuse are desperately needed to maintain muscle mass. We recently found that massage as a mechanotherapy can improve muscle regrowth following disuse atrophy. Therefore, we aimed to determine if massage has similar anabolic effects when applied during normal weight bearing conditions (WB) or during atrophy induced by hindlimb suspension (HS) in adult rats. Adult (10months) male Fischer344-Brown Norway rats underwent either hindlimb suspension (HS, n=8) or normal WB (WB, n=8) for 7days. Massage was applied using cyclic compressive loading (CCL) in WB (WBM, n=9) or HS rats (HSM, n=9) and included four 30-minute bouts of CCL applied to gastrocnemius muscle every other day. Massage had no effect on any anabolic parameter measured under WB conditions (WBM). In contrast, massage during HS (HSM) stimulated protein turnover, but did not mitigate muscle atrophy. Atrophy from HS was caused by both lowered protein synthesis and higher degradation. HS and HSM had lowered total RNA compared with WB and this was the result of significantly higher ribosome degradation in HS that was attenuated in HSM, without differences in ribosomal biogenesis. Also, massage increased protein turnover in the non-massaged contralateral limb during HS. Finally, we determined that total RNA degradation primarily dictates loss of muscle ribosomal content during disuse atrophy. We conclude that massage is an effective mechanotherapy to impact protein turnover during muscle disuse in both the massaged and non-massaged contralateral muscle, but it does not attenuate the loss of muscle mass.

Mitochondrial Metabolism in Cancer Cachexia: Novel Drug Target.

Cancer cachexia is a metabolic syndrome prevalent in the majority of the advanced cancers and is associated with complications such as anorexia, early satiety, weakness, anaemia, and edema, thereby reducing performance and impairing quality of life. Skeletal muscle wasting is a characteristic feature of cancer-cachexia and mitochondria is responsible for regulating total protein turnover in skeletal muscle tissue. We carried out exhaustive search for cancer cachexia and role of mitochondria in the same in various databases. All the relevant articles were gathered and the pertinent information was extracted out and compiled which was further structured into different sub-sections. Various findings on the mitochondrial alterations in connection to its disturbed normal physiology in various models of cancer-cachexia have been recently reported, suggesting a significant role of the organelle in the pathogenesis of the complications involved in the disorder. It has also been reported that reduced mitochondrial oxidative capacity is due to reduced mitochondrial biogenesis as well as altered balance between fusion and fission protein activities. Moreover, autophagy in mitochondria (termed as mitophagy) is reported to play an important role in cancer cachexia. The present review aims to put forth the changes occurring in mitochondria and hence explore possible targets which can be exploited in cancer-induced cachexia for treatment of such a debilitating condition.

Leucine Improved Growth Performance, Muscle Growth, and Muscle Protein Deposition Through AKT/TOR and AKT/FOXO3a Signaling Pathways in Hybrid Catfish Pelteobagrus vachelli × Leiocassis longirostris.

(1) Background: l-leucine (Leu) plays a positive role in regulating protein turnover in skeletal muscle in mammal. However, the molecular mechanism for the effects of Leu on muscle growth and protein deposition is not clearly demonstrated in fish. This study investigated the effects of dietary Leu on growth performance and muscle growth, protein synthesis, and degradation-related signaling pathways of hybrid catfish (Pelteobagrus vachelli♀ × Leiocassis longirostris♂). (2) Methods: A total of 630 hybrid catfish (23.19 ± 0.20 g) were fed 6 different experimental diets containing graded levels of Leu at 10.0 (control), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g Leu kg-1 for 8 weeks. (3) Results: Results showed that dietary Leu increased percent weight gain (PWG), specific growth rate (SGR), FI (feed intake), feed efficiency (FE), protein efficiency ratio (PER), muscle fibers diameter, and muscle fibers density; up-regulated insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), proliferating cell nuclear antigen (PCNA), myogenic regulation factors (MyoD, Myf5, MyoG, and Mrf4), and MyHC mRNA levels; increased muscle protein synthesis via regulating the AKT/TOR signaling pathway; and attenuated protein degradation via regulating the AKT/FOXO3a signaling pathway. (4) Conclusions: These results suggest that Leu has potential role to improve muscle growth and protein deposition in fish, which might be due to the regulation of IGF mRNA expression, muscle growth related gene, and protein synthesis and degradation-related signaling pathways. Based on the broken-line model, the Leu requirement of hybrid catfish (23.19-54.55 g) for PWG was estimated to be 28.10 g kg-1 of the diet (73.04 g kg-1 of dietary protein). These results will improve our understanding of the mechanisms responsible for muscle growth and protein deposition effects of Leu in fish.

The Influence of Omega-3 Fatty Acids on Skeletal Muscle Protein Turnover in Health, Disuse, and Disease.

Ingestion of omega-3 fatty acids is known to exert favorable health effects on a number of biological processes such as improved immune profile, enhanced cognition, and optimized neuromuscular function. Recently, data have emerged demonstrating a positive influence of omega-3 fatty acid intake on skeletal muscle. For instance, there are reports of clinically-relevant gains in muscle size and strength in healthy older persons with omega-3 fatty acid intake as well as evidence that omega-3 fatty acid ingestion alleviates the loss of muscle mass and prevents decrements in mitochondrial respiration during periods of muscle-disuse. Cancer cachexia that is characterized by a rapid involuntary loss of lean mass may also be attenuated by omega-3 fatty acid provision. The primary means by which omega-3 fatty acids positively impact skeletal muscle mass is via incorporation of eicosapentaenoic acid (EPA; 20:5n−3) and docosahexaenoic acid (DHA; 22:6n−3) into membrane phospholipids of the sarcolemma and intracellular organelles. Enrichment of EPA and DHA in these membrane phospholipids is linked to enhanced rates of muscle protein synthesis, decreased expression of factors that regulate muscle protein breakdown, and improved mitochondrial respiration kinetics. However, exactly how incorporation of EPA and DHA into phospholipid membranes alters these processes remains unknown. In this review, we discuss the interaction between omega-3 fatty acid ingestion and skeletal muscle protein turnover in response to nutrient provision in younger and older adults. Additionally, we examine the role of omega-3 fatty acid supplementation in protecting muscle loss during muscle-disuse and in cancer cachexia, and critically evaluate the molecular mechanisms that underpin the phenotypic changes observed in skeletal muscle with omega-3 fatty acid intake.

Endogenous CSE/Hydrogen Sulfide System Regulates the Effects of Glucocorticoids and Insulin on Muscle Protein Synthesis.

Aims Insulin and glucocorticoids play crucial roles in skeletal muscle protein turnover. Fast-twitch glycolytic fibres are more susceptible to atrophy than slow-twitch oxidative fibres. Based on accumulating evidence, hydrogen sulfide (H2S) is a physiological mediator of this process. The regulatory effect of H2S on protein synthesis in fast-twitch fibres was evaluated. Results A NaHS (sodium hydrosulfide) injection simultaneously increased the diameter of M. pectoralis major (i.e., fast-twitch glycolytic fibres) and activated the mammalian target of the rapamycin (mTOR)/p70S6 kinase (p70S6K) pathway. Dexamethasone (DEX) inhibited protein synthesis, downregulated mTOR and p70S6K phosphorylation, and suppressed the expression of the cystathionine γ-lyase (CSE) protein in myoblasts. The precursor of H2S, L-cysteine, completely abolished the inhibitory effects of DEX. The CSE inhibitor DL-propargylglycine (PAG) completely abrogated the effects of RU486 on blocking the suppressive effects of DEX. The H2S donor NaHS increased the H2S concentrations and abrogated the inhibitory effects of DEX on protein synthesis. Insulin increased protein synthesis and upregulated CSE expression. However, PAG abrogated the stimulatory effects of insulin on protein synthesis and the activity of the mTOR/p70S6K pathway. Innovation These results demonstrated that CSE/H2S regulated protein synthesis in fast-twitch muscle fibres, and glucocorticoids and insulin regulated protein synthesis in an endogenous CSE/H2S system-dependent manner. Conclusions The results from the present study suggest that the endogenous CSE/H2S system regulates fast-twitch glycolytic muscle degeneration and regeneration.

Mediators of cachexia in cancer patients.

Alterations in amino acid and protein metabolism-particularly in skeletal muscle-are a key feature of cancer that contributes to the cachexia syndrome. Thus, skeletal muscle protein turnover is characterized by an exacerbated rate of protein degradation, promoted by an activation of different proteolytic systems that include the ubiquitin-proteasome and the autophagic-lysosomal pathways. These changes are promoted by both hormonal alterations and inflammatory mediators released as a result of the systemic inflammatory response induced by the tumor. Other events, such as alterations in the rate of myogenesis/apoptosis and decreased regeneration potential also affect skeletal muscle in patients with cancer. Mitochondrial dysfunction also contributes to changes in skeletal muscle metabolism and further contributes to the exacerbation of the cancer-wasting syndrome. Different inflammatory mediators-either released by the tumor or by the patient's healthy cells-are responsible for the activation of these catabolic processes that take place in skeletal muscle and in other tissues/organs, such as liver or adipose tissues. Indeed, white adipose tissue is also subject to extensive wasting and "browning" of some of the white adipocytes into beige cells; therefore increasing the energetic inefficiency of the patient with cancer. Recently, an interest in the role of micromRNAs-either free or transported into exosomes-has been related to the events that take place in white adipose tissue during cancer cachexia.

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL– , high-fat oil– (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.

Reduced Skeletal Muscle Protein Turnover and Thyroid Hormone Metabolism in Adaptive Thermogenesis That Facilitates Body Fat Recovery During Weight Regain.

Objective: The recovery of body composition after weight loss is characterized by an accelerated rate of fat recovery (preferential catch-up fat) resulting partly from an adaptive suppression of thermogenesis. Although the skeletal muscle has been implicated as an effector site for such thrifty (energy conservation) metabolism driving catch-up fat, the underlying mechanisms remain to be elucidated. We test here the hypothesis that this thrifty metabolism driving catch-up fat could reside in a reduced rate of protein turnover (an energetically costly “futile” cycle) and in altered local thyroid hormone metabolism in skeletal muscle.Methods: Using a validated rat model of semistarvation-refeeding in which catch-up fat is driven solely by suppressed thermogenesis, we measured after 1 week of refeeding in refed and control animals the following: (i) in-vivo rates of protein synthesis in hindlimb skeletal muscles using the flooding dose technique of 13C-labeled valine incorporation in muscle protein, (ii) ex-vivo muscle assay of net formation of thyroid hormone tri-iodothyronine (T3) from precursor hormone thyroxine (T4), and (iii) protein expression of skeletal muscle deiodinases (type 1, 2, and 3).Results: We show that after 1 week of calorie-controlled refeeding, the fractional protein synthesis rate was lower in skeletal muscles of refed animals than in controls (by 30–35%, p < 0.01) despite no between-group differences in the rate of skeletal muscle growth or whole-body protein deposition—thereby underscoring concomitant reductions in both protein synthesis and protein degradation rates in skeletal muscles of refed animals compared to controls. These differences in skeletal muscle protein turnover during catch-up fat were found to be independent of muscle type and fiber composition, and were associated with a slower net formation of muscle T3 from precursor hormone T4, together with increases in muscle protein expression of deiodinases which convert T4 and T3 to inactive forms.Conclusions: These results suggest that diminished skeletal muscle protein turnover, together with altered local muscle metabolism of thyroid hormones leading to diminished intracellular T3 availability, are features of the thrifty metabolism that drives the rapid restoration of the fat reserves during weight regain after caloric restriction.

Protein imbalance in the development of skeletal muscle wasting in tumour-bearing mice.

BackgroundCancer cachexia occurs in approximately 80% of cancer patients and is a key contributor to cancer‐related death. The mechanisms controlling development of tumour‐induced muscle wasting are not fully elucidated. Specifically, the progression and development of cancer cachexia are underexplored. Therefore, we examined skeletal muscle protein turnover throughout the development of cancer cachexia in tumour‐bearing mice.MethodsLewis lung carcinoma (LLC) was injected into the hind flank of C57BL6/J mice at 8 weeks age with tumour allowed to develop for 1, 2, 3, or 4 weeks and compared with PBS injected control. Muscle size was measured by cross‐sectional area analysis of haematoxylin and eosin stained tibialis anterior muscle. 2H2O was used to assess protein synthesis throughout the development of cancer cachexia. Immunoblot and RT‐qPCR were used to measure regulators of protein turnover. TUNEL staining was utilized to measure apoptotic nuclei. LLC conditioned media (LCM) treatment of C2C12 myotubes was used to analyse cancer cachexia in vitro.ResultsMuscle cross‐sectional area decreased ~40% 4 weeks following tumour implantation. Myogenic signalling was suppressed in tumour‐bearing mice as soon as 1 week following tumour implantation, including lower mRNA contents of Pax7, MyoD, CyclinD1, and Myogenin, when compared with control animals. AchRδ and AchRε mRNA contents were down‐regulated by ~50% 3 weeks following tumour implantation. Mixed fractional synthesis rate protein synthesis was ~40% lower in 4 week tumour‐bearing mice when compared with PBS controls. Protein ubiquitination was elevated by ~50% 4 weeks after tumour implantation. Moreover, there was an increase in autophagy machinery after 4 weeks of tumour growth. Finally, ERK and p38 MAPK phosphorylations were fourfold and threefold greater than control muscle 4 weeks following tumour implantation, respectively. Inhibition of p38 MAPK, but not ERK MAPK, in vitro partially rescued LCM‐induced loss of myotube diameter.ConclusionsOur findings work towards understanding the pathophysiological signalling in skeletal muscle in the initial development of cancer cachexia. Shortly following the onset of the tumour‐bearing state alterations in myogenic regulatory factors are apparent, suggesting early onset alterations in the capacity for myogenic induction. Cancer cachexia presents with a combination of a loss of protein synthesis and increased markers of protein breakdown, specifically in the ubiquitin‐proteasome system. Also, p38 MAPK may be a potential therapeutic target to combat cancer cachexia via a p38‐FOX01‐atrogene‐ubiquitin‐proteasome mechanism.

Hypoxia impairs adaptation of skeletal muscle protein turnover- and AMPK signaling during fasting-induced muscle atrophy

BackgroundHypoxemia in humans may occur during high altitude mountaineering and in patients suffering from ventilatory insufficiencies such as cardiovascular- or respiratory disease including Chronic Obstructive Pulmonary Disease (COPD). In these conditions, hypoxemia has been correlated to reduced appetite and decreased food intake. Since hypoxemia and reduced food intake intersect in various physiological and pathological conditions and both induce loss of muscle mass, we investigated whether hypoxia aggravates fasting-induced skeletal muscle atrophy and evaluated underlying protein turnover signaling.MethodsMice were kept under hypoxic (8% oxygen) or normoxic conditions (21% oxygen), or were pair-fed to the hypoxia group for 12 days. Following an additional 24 hours of fasting, muscle weight and protein turnover signaling were assessed in the gastrocnemius muscle by RT-qPCR and Western blotting.ResultsLoss of gastrocnemius muscle mass in response to fasting in the hypoxic group was increased compared to the normoxic group, but not to the pair-fed normoxic control group. Conversely, the fasting-induced increase in poly-ubiquitin conjugation, and expression of the ubiquitin 26S-proteasome E3 ligases, autophagy-lysosomal degradation-related mRNA transcripts and proteins, and markers of the integrated stress response (ISR), were attenuated in the hypoxia group compared to the pair-fed group. Mammalian target of rapamycin complex 1 (mTORC1) downstream signaling was reduced by fasting under normoxic conditions, but sustained under hypoxic conditions. Activation of AMP-activated protein kinase (AMPK) / tuberous sclerosis complex 2 (TSC2) signaling by fasting was absent, in line with retained mTORC1 activity under hypoxic conditions. Similarly, hypoxia suppressed AMPK-mediated glucocorticoid receptor (GR) signaling following fasting, which corresponded with blunted proteolytic signaling responses.ConclusionsHypoxia aggravates fasting-induced muscle wasting, and suppresses AMPK and ISR activation. Altered AMPK-mediated regulation of mTORC1 and GR may underlie aberrant protein turnover signaling and affect muscle atrophy responses in hypoxic skeletal muscle.

Recent advances in understanding the role of FOXO3.

The forkhead box O3 (FOXO3, or FKHRL1) protein is a member of the FOXO subclass of transcription factors. FOXO proteins were originally identified as regulators of insulin-related genes; however, they are now established regulators of genes involved in vital biological processes, including substrate metabolism, protein turnover, cell survival, and cell death. FOXO3 is one of the rare genes that have been consistently linked to longevity in in vivo models. This review provides an update of the most recent research pertaining to the role of FOXO3 in (i) the regulation of protein turnover in skeletal muscle, the largest protein pool of the body, and (ii) the genetic basis of longevity. Finally, it examines (iii) the role of microRNAs in the regulation of FOXO3 and its impact on the regulation of the cell cycle.

Beta2 -adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men.

Animal models have shown that beta2 -adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2 -adrenoceptor is a potential target in the treatment of disuse-, disease- and age-related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2 -adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A-signalling, activation of Akt2 and modulation of mRNA levels of growth-regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2 -adrenoceptor agonist treatment during recovery from resistance exercise in humans. The effect of beta2 -adrenoceptor stimulation on skeletal muscle protein turnover and intracellular signalling is insufficiently explored in humans, particularly in association with exercise. In a randomized, placebo-controlled, cross-over study investigating 12 trained men, the effects of beta2 -agonist (6×4mg oral salbutamol) on protein turnover rates, intracellular signalling and mRNA response in skeletal muscle were investigated 0.5-5h after quadriceps resistance exercise. Each trial was preceded by a 4-day lead-in treatment period. Leg protein turnover rates were assessed by infusion of [13 C6 ]-phenylalanine and sampling of arterial and venous blood, as well as vastus lateralis muscle biopsies 0.5 and 5h after exercise. Furthermore, myofibrillar fractional synthesis rate, intracellular signalling and mRNA response were measured in muscle biopsies. The mean (95% confidence interval) myofibrillar fractional synthesis rate was higher for salbutamol than placebo [0.079(95% CI, 0.064 to 0.093) vs. 0.066(95% CI, 0.056 to 0.075%)×h-1 ] (P<0.05). Mean net leg phenylalanine balance 0.5-5h after exercise was higher for salbutamol than placebo [3.6(95% CI, 1.0 to 6.2nmol)×min-1 ×100 gLeg Lean Mass-1 ] (P<0.01). Phosphorylation of Akt2, cAMP response element binding protein and PKA substrate 0.5 and 5h after exercise, as well as phosphorylation of eEF2 5h after exercise, was higher (P<0.05) for salbutamol than placebo. Calpain-1, Forkhead box protein O1, myostatin and Smad3 mRNA content was higher (P<0.01) for salbutamol than placebo 0.5h after exercise, as well as Forkhead box protein O1 and myostatin mRNA content 5h after exercise, whereas ActivinRIIB mRNA content was lower (P<0.01) for salbutamol 5h after exercise. These observations suggest that beta2 -agonist increases protein turnover rates in skeletal muscle after resistance exercise in humans, with concomitant cAMP/PKA and Akt2 signalling, as well as modulation of mRNA response of growth-regulating proteins.