Reduced Muscle Protein Synthesis Research Articles

Precocious glucocorticoid exposure reduces skeletal muscle satellite cells in the fetal rat.

Perinatal skeletal muscle growth rates are a function of protein and myonuclear accretion. Precocious exposure of the fetus to glucocorticoids (GLC) in utero impairs muscle growth. Reduced muscle protein synthesis rates contribute to this response, but the consequences for myonuclear hyperplasia are unknown. To test the hypothesis that blunting of Pax7+ muscle progenitor cell proliferative activity by GLC in vivo also contributes to reduced fetal muscle growth, pregnant rats were administered dexamethasone (DEX: 1 mg/L drinking water) from embryonic day (ED) 13 to ED21. Their responses were compared to pair-fed (PF) and ad libitum-fed controls (CON). Bromodeoxyuridine (BrdU) was administered before delivery to measure myonuclear accretion. Fetal hind limb and diaphragm muscles were collected at term and analyzed for myofiber cross-sectional area (CSA), total and BrdU+ myonuclei, Pax7+ nuclei, MyoD and myogenin protein and mRNA abundance and myosin heavy chain (MyHC) isoform composition. Mean fiber CSA, myonuclei/myofiber and Pax7+ nuclei/myofiber ratios were reduced in DEX compared to those in CON and PF muscles; CSA/myonucleus, BrdU+/total myonuclei and BrdU+ myonuclei/Pax7+ nuclei were similar among groups. Myogenin abundance was reduced and MyHC-slow was increased in DEX fetuses. The data are consistent with GLC inhibition of muscle progenitor cell proliferation limiting satellite cell and myonuclear accretion. The response of PF-fed compared to CON muscles indicated that decreased food consumption by DEX dams contributed to the smaller myofiber CSA but did not affect Pax7+ nuclear accretion. Thus, the effect on satellite cell reserve and myonuclear number also contributes to the blunting of fetal muscle growth by GLC.

Leucine ameliorates endotoxin‐induced alterations in protein‐protein interactions within mTORC1 complex in neonatal piglets

Sepsis decreases protein synthesis in skeletalmuscle in part by limiting the phosphorylation of translation initiationregulatory factors, eukaryotic initiation factor (eIF) 4E‐binding protein (4EBP1)and S6 kinase (S6K1), that are downstream of the mammalian target of rapamycincomplex 1 (mTORC1). Previous work inmature rats suggests that decreased raptor binding activity to eIF3b mayunderline the sepsis‐induced reduction in protein synthesis in skeletal muscle. Raptor‐eIF3b interaction is crucial for thestimulation of protein translation and its alteration during endotoxemia inmature animals appears to block the anabolic response to the branched‐chainamino acid, leucine (Leu). We have shown in healthy neonatal pigs that Leu supplementation increases muscle protein synthesis by enhancing the activation of mTORC1 and its downstream targets, 4EBP1 and S6K1. To determine if supplemental Leu can stimulate raptor‐eIF3b binding and muscle protein synthesis in theneonate during endotoxemia, overnight fasted neonatal pigs (n=6–8/group) were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic‐substrate clamps. Leu (400 μmol·kg−1·hr−1) or saline was infused during the last h. Phosphorylation of 4EBP1 and S6K1, eIF4E‐eIF4G complex formation, raptor‐eIF3b binding, protein synthesis rates, ribosomal translational capacity (Cs) and translational efficiency (KRNA) were determined in longissimus dorsi (LD), gastrocnemius (Gast) and soleus muscles. LPS decreased protein synthesis and KRNA in LD, Gast, and soleus muscles (P<0.05) while Cs did not change compared to controls. Leu infusion in the LPS group increased 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G complex formation, KRNA, and the binding of raptor to eIF3b (by 2 to 3 fold) and protein synthesis (by over 80%) in LD, Gast, and soleus muscles compared to LPS infusion alone (P<0.05). Compared to saline, Leu infusion increased protein synthesis, KRNA, raptor‐eIF3b interaction, 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles (P<0.05). Raptor‐eIF3b binding showed a linear correlation with 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G formation, and protein synthesis in LD, Gast, and soleus muscles that was independent of treatment(P<0.01). These findings suggest that parenteral Leu supplementation attenuates the reduction in muscle protein synthesis induced by endotoxemia by stimulating the interaction of raptor with eIF3 within the mTORC1 complex in the neonate.Support or Funding InformationSupported by NIH AR444474, USDA/ARS 6250‐51000‐055, NIH HD072891, and USDA NIFA 2013‐67015‐20438

Nucleoprotein‐enriched diet prevents disuse‐induced muscle atrophy in soleus muscle of rats

Disuse leads to a decrease of mass, fiber cross‐sectional area, protein synthesis, and satellite cell activity in skeletal muscle. It is known that the depletion of amino acids reduces muscle protein synthesis. In addition, nucleotides affect cell proliferation and differentiation. Thus, amino acids and nucleotides are essential nutrition for protein synthesis, cell proliferation, and differentiation in skeletal muscle. Therefore, the present study investigated the effect of nucleoprotein, which has combined amino acids and nucleotides, enriched diet during unloading on protein synthesis, activity of satellite cell, and muscle atrophy. Female Wistar rats were assigned randomly into 4 groups: control group (CON), control with nucleoprotein group (CON+NP), hindlimb unloading (HU), and hindlimb unloading with nucleoprotein group (HU+NP). Fiber cross‐sectional area (FCSA) was significantly higher in HU+NP group than HU group although unloading resulted in a decrease of CSA. In addition, the levels of phosphorylated‐ribosomal protein S6 (rpS6) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were significantly higher in HU+NP group than HU group, whereas the levels of phosphorylated‐p70S6 kinase (p70S6K) were equivalent among these groups. Furthermore, the numbers of MyoD and myogenin‐positive nuclei cells were significantly higher in HU+NP group than HU group. These results suggest that nucleoprotein exerts preventive effects on muscle atrophy via activations of rpS6 and satellite cells, which are regulated with ERK signaling independently from p70S6K pathway.Support or Funding InformationSupported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and the internal funds of Nissan Chemical Industries, Ltd.

Role of insulin in the regulation of human skeletal muscle protein synthesis and breakdown: a systematic review and meta-analysis.

We aimed to investigate the role of insulin in regulating human skeletal muscle metabolism in health and diabetes. We conducted a systematic review and meta-analysis of published data that examined changes in skeletal muscle protein synthesis (MPS) and/or muscle protein breakdown (MPB) in response to insulin infusion. Random-effects models were used to calculate weighted mean differences (WMDs), 95% CIs and corresponding p values. Both MPS and MPB are reported in units of nmol (100ml leg vol.)(-1) min(-1). A total of 104 articles were examined in detail. Of these, 44 and 25 studies (including a total of 173 individuals) were included in the systematic review and meta-analysis, respectively. In the overall estimate, insulin did not affect MPS (WMD 3.90 [95% CI -0.74, 8.55], p = 0.71), but significantly reduced MPB (WMD -15.46 [95% CI -19.74, -11.18], p < 0.001). Overall, insulin significantly increased net balance protein acquisition (WMD 20.09 [95% CI 15.93, 24.26], p < 0.001). Subgroup analysis of the effect of insulin on MPS according to amino acid (AA) delivery was performed using meta-regression analysis. The estimate size (WMD) was significantly different between subgroups based on AA availability (p = 0.001). An increase in MPS was observed when AA availability increased (WMD 13.44 [95% CI 4.07, 22.81], p < 0.01), but not when AA availability was reduced or unchanged. In individuals with diabetes and in the presence of maintained delivery of AA, there was a significant reduction in MPS in response to insulin (WMD -6.67 [95% CI -12.29, -0.66], p < 0.05). This study demonstrates the complex role of insulin in regulating skeletal muscle metabolism. Insulin appears to have a permissive role in MPS in the presence of elevated AAs, and plays a clear role in reducing MPB independent of AA availability.

Leucine Attenuates the Endotoxin‐induced Reduction in Skeletal Muscle Protein Synthesis in Neonatal Pigs

Sepsis decreases protein synthesis (PS) in skeletal muscle and mitigates the anabolic response to Leucine (Leu) in mature animals. To determine if supplemental Leu can stimulate muscle PS in the neonate during endotoxemia, overnight fasted neonatal pigs were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic substrate clamps. Leu or saline was infused during the last h. PS and translation initiation factor activation were determined in skeletal muscle. LPS decreased protein synthesis by 36% in longissimus dorsi (LD), 28% in gastrocnemius (Gast) and 38% in soleus muscles compared to controls, but Leu infusion in the LPS group increased protein synthesis by 84% in LD, 81% in Gast and 83% in soleus compared to LPS infusion alone. Leu infusion alone compared to controls also increased protein synthesis in the LD, Gast and Soleus muscles. Leu infusion in both healthy and endotoxemic piglets compared to non-Leu infused piglets increased 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles. These findings suggest that Leu supplementation can attenuate the reduction in muscle PS induced by endotoxemia by stimulating mTORC1-dependent translation in the neonate. (Supported by NIH AR444474, USDA/ARS 6250-51000-055, NIH HD072891, and USDA NIFA 2013-67015-20438)

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

Myostatin signaling regulates Akt activity via the regulation of miR-486 expression

Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

Could vitamin D and bicarbonate supplementation synergize to mitigate age-related loss of muscle?

In patients with kidney disease receiving hemodialysis, metabolic acidosis is closely tied to muscle pathology and is known to exacerbate age-related loss of muscle mass and function. To a lesser extent, the ‘‘Western’’ diet is thought to contribute to mild (sub-clinical) metabolic acidosis via the supply of acid precursors from foods [1], but the clinical significance to chronic age-related muscle degeneration has received little attention. This may partly be due to equivocal data on the benefits of neutralizing diet-induced metabolic acidosis for improving muscle-related outcomes in patients without kidney disease [2]. Despite limited data, acidosis has been shown to stimulate muscle proteolysis and is associated with negative functional outcomes including reduced peak torque and lower gait speed [3]. This is important to note because aged adults are less able to excrete excess H ions and are more prone to develop mild, but slowly increasing metabolic acidosis that may be exacerbated by a ‘‘Western’’ diet [1]. Furthermore, approximately one out of four adults older than 50 years of age have low serum bicarbonate levels to neutralize an acidogenic diet. Because of these factors, recommendations for future work include examining the effects of alkali therapy on functional outcomes, nitrogen balance, strength and other relevant measures of physical function, in aged healthy adults [3]. Vitamin D status (measured as 25(OH)D) is also garnering interest as a player in preserving skeletal muscle mass and function in aging. Although, the study of vitamin D deficiency and repletion, in relation to muscle metabolic function in aging is relatively new, vitamin D deficiency has already been associated with several negative muscle outcomes including muscle degeneration with fat infiltration [4]. Loss of muscle fiber size and reduction in muscle protein synthesis have also been documented and is thought to be clinically significant since vitamin D deficiency is considered as a worldwide epidemic [5]. In this issue of Endocrine, Ceglia et al. [6] investigated the interactions between potassium bicarbonate and vitamin D supplementation to shed light on the underlying mechanisms associated with muscle atrophy, conservation, and fiber morphology in male rats. The author’s rationale for examining the vitamin D/bicarbonate interaction was based on research from the 1970s citing that depletion of vitamin D resulted in metabolic acidosis, whereas repletion resulted in a metabolic alkalosis by changes in renal tubule bicarbonate reabsorption. An additional cited study provided support that acute metabolic acidosis resulted in suppression of 1-alpha hydroxylase activity, collectively suggesting that the alterations in either vitamin D status or acid–base balance may affect the other and, thus, have detrimental impact on muscle tissue [6]. Ceglia et al. [6] found that potassium bicarbonate supplementation resulted in 35 % lower urinary nitrogen to creatinine (UNi/Cr) ratio independent of vitamin D status. In vitamin D deficient rats, potassium bicarbonate resulted in 28 % increase in UNi/Cr compared to rats with normal vitamin D levels, but the findings did not reach the statistical significance. Furthermore, in rats experiencing metabolic acidosis, higher vitamin D status seemed to work synergistically with bicarbonate to potentiate Akt activation, a protein kinase involved in several anabolic pathways. As a result of their findings, the authors suggested that longer more comprehensive investigation is required to advance and confirm the impact of a dietary intervention on ubiquitin–proteasome pathway at different pathway time points and to fully D. Travis Thomas (&) Division of Clinical Nutrition, University of Kentucky, Lexington, KY, USA e-mail: david.t.thomas@uky.edu

Muscle recovery after immobilization is improved by dietary supplementation with alpha‐hydroxyisocaproic acid (HICA)

We posited that αHICA, a leucine metabolite, will slow the loss or improve recovery of muscle mass in response to disuse. Adult rats were fed a diet supplemented with αHICA or an isocaloric isonitrogenous control diet. Disuse was produced by unilateral hindlimb immobilization (casting) for 7 d, using the contralateral muscle as control, or the cast removed and muscle recovered for 14 d. Casting decreased gastrocnemius mass which was associated with reduced muscle protein synthesis (MPS) and S6K1 phosphorylation, but increased proteasome activity and atrogin‐1 and MuRF1 mRNA. αHICA did not prevent casting‐induced muscle atrophy, but the typical decrement in MPS was not detected. αHICA did not alter the immobilization‐induced increase in proteasome activity and atrogene expression. After 14 d of recovery, muscle mass had returned to control values only in αHICA‐fed rats and this was associated with increased MPS and S6K1 and 4EBP1 phosphorylation in previously immobilized muscle. Proteasome activity and atrogene content remained at control levels during recovery and were not affected by αHICA. Our data suggest αHICA supplementation does not slow muscle loss produced by disuse, but does speed recovery in part by maintaining increased MPS.

Effects of malnutrition with or without eicosapentaenoic acid on proteolytic pathways in diaphragm

Attenuation of muscle wasting has been reported with eicosapentaenoic acid (EPA) use in cachectic states. Pathways mediating muscle proteolysis with severe short-term nutritional deprivation (ND)±EPA were evaluated, including diaphragm fiber-specific cross-sectional areas, mRNA (real-time PCR) and protein expression (Western blot). Rats were divided into three groups: (1) free-eating controls, (2) ND and (3) ND+EPA. ND significantly influenced multiple proteolytic pathways. EPA significantly reduced mRNA abundances for most genes to control levels with ND. However, discordant muscle protein expression of many genes was noted with the use of EPA, as protein levels failed to fall. EPA had no impact on diaphragm muscle atrophy, despite the impressive mRNA and some protein results. We conclude that EPA does not attenuate diaphragm muscle atrophy with severe levels of ND. Postulated mechanisms include reduction in muscle protein synthesis and persistent ongoing stimuli for proteolysis. Our study provides unique data on proteolytic signals with ND and has important implications for future studies using EPA.

The biochemical actions of phentolamine and papaverine on rat perfused skeletal muscle.

The direct actions of the vasodilators, papaverine and phentolamine, on skeletal muscle metabolism were investigated in an isolated perfusion system. Eviscerated male rats were hemisected above the diaphragm and perfused, via the aorta, with a physiological perfusion medium containing erythrocytes. Papaverine, but not phentolamine, reduced vascular resistance throughout the 80 min study. Papaverine caused marked reductions in muscle concentrations of ATP and phosphocreatine, when compared with muscle from preparations without added vasodilators. This was accompanied by elevations in lactate concentrations. Water content of papaverine-treated muscle was also higher than values in unperfused muscle taken in-vivo. Phentolamine, in contrast, had no effect on muscle ATP, phosphocreatine, lactate or water content. The metabolism of the entire preparation was also investigated. Papaverine induced increases in lactate output while phentolamine treatment caused an initial uptake, followed by an increased output of lactate. There was no significant effect of either papaverine or phentolamine on the metabolism of K+ and glucose. Arteriovenous differences in oxygen-saturation of haemoglobin and pH were also unaltered. Investigations on aspects of protein metabolism demonstrated that papaverine and phentolamine caused significant reductions in muscle protein synthesis when compared with control perfusions or in-vivo values. The reductions in synthesis were not due to reductions in cAMP or limitations in branched-chain amino acid supply. However, there was the suggestion that phentolamine caused a decrease in protein breakdown. The overall data indicated that papaverine and phentolamine may cause impairment of skeletal muscle metabolism. This has important implications for their therapeutic or experimental use.

Non‐steroidal anti‐inflammatory drug (NSAID) administration to mature and old horses influences the activation of translation initiation factors

Aging is associated with chronic inflammation, which has been associated with reduced muscle protein synthesis in other species. In humans and rodents, muscle mass loss associated with aging has been linked to reductions in phosphorylation (P) of translation initiation factors (eIF) in response to anabolic stimuli; however, this has not been studied in horses. Thus, the objectives of this study were to characterize the differences in P of eIF between old and mature horses, and to determine whether P of eIF was affected by 2 and 4 wk of administration of a NSAID, phenylbutazone (2 g/day). Muscle biopsies were obtained from the gluteus medius of 6 mature (7–14 y old) and 6 old (21–26 y old) horses following 6 h of intermittent small meal feeding for the measurement of P of eIF (Akt at Ser473, S6 Kinase at Thr389, rpS6 at Ser235/236and240/244, and 4EBP1 at Thr37/46) using Western blotting. S6 Kinase P-Thr389 was significantly affected by time on NSAID (P= 0.008), age (P= 0.05), and age*time (P= 0.05). There was no effect of age (P > 0.05) on P of the other eIF; however, there was a significant increase in Akt P-Ser473 (P= 0.007), and trend to increase rpS6 P-Ser235/236&240/244 (P= 0.08) with length of time on NSAID. There were no changes in 4EBP1 P-Thr37/46. The potential reduction in inflammation in old horses due to NSAID administration did not consistently stimulate P of eIF, possibly because horses were fed small meals intermittently rather than receiving a single large meal. Additional research, using isotope techniques, is necessary to determine whether increases in Akt, S6 Kinase, and rpS6 P corresponded to increases in protein synthesis. Grant Funding Source: Morris Animal Foundation

Sepsis and Mechanical Ventilation Restrain Translation Initiation in Skeletal Muscle by Inducing AMPK‐associated TSC2 Restriction of mTOR Signaling in Pigs

In skeletal muscle, AMP‐activated protein kinase (AMPK) acts as a cellular energy sensor of AMP:ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)‐induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothesize that mechanical ventilation and sepsis restrain translation in skeletal muscle by activation of AMPK. Neonatal pigs (n=8/group) were subjected to mechanical ventilation for 9 h in the absence (MV) and presence of LPS (LPSMV). AMPK and translation activation signals were determined in longissimus dorsi muscle. Plasma insulin levels rose with MV and were greatest in LPSMV pigs. In muscle, despite higher PKB activation, MV and LPS additively reduced mTOR phosphorylation and decreased the phosphorylation of tuberous sclerosis complex 2 (TSC2) on Thr1462. AMPK phosphorylation increased during MV and was augmented further in LPSMV pigs. TSC2 phosphorylation on the AMPK site, Ser1387, increased in MVLPS. MV decreased eIF4G·eIF4E association, which was further decreased by LPS. These findings suggest that both MV and sepsis increase AMPK activation in peripheral skeletal muscle, leading to inhibition of TSC2‐mediated mTOR‐dependent translation signaling when MV and sepsis coexist. NIH AR51563, NIH AR44474, and USDA 6250‐51000‐040.

Mechanisms regulating muscle mass during disuse atrophy and rehabilitation in humans.

Muscle mass loss accompanies periods of bedrest and limb immobilization in humans and requires rehabilitation exercise to effectively restore mass and function. Although recent evidence points to an early and transient rise in muscle protein breakdown contributing to this decline in muscle mass, the driving factor seems to be a reduction in muscle protein synthesis, not least in part due to the development of anabolic resistance to amino acid provision. Although the AKT signaling pathway has been identified in small animals as central to the regulation of muscle protein synthesis, several studies in humans have now demonstrated a disassociation between AKT signaling and muscle protein synthesis during feeding, exercise, and immobilization, suggesting that the mechanisms regulating protein synthesis in human skeletal muscle are more complex than initially thought (at least in non-inflammatory states). During rehabilitation, exercise-induced myogenesis may in part be responsible for the recovery of muscle mass. Rapid and sustained exercise-induced suppression of myostatin mRNA expression, that precedes any gain in muscle mass, points to this, along with other myogenic proteins, as being potential regulators of muscle regeneration during exercise rehabilitation in humans.

Development of a Technique for Introduction of an Expressed cDNA into Parathyroid Cells by Direct Injection

Development of a Technique for Introduction of an Expressed cDNA into Parathyroid Cells by Direct Injection

Effects of a Novel Selective Androgen Receptor Modulator on Dexamethasone-Induced and Hypogonadism-Induced Muscle Atrophy

Glucocorticoids are the most widely used antiinflammatory drugs in the world. However, prolonged use of glucocorticoids results in undesirable side effects such as muscle wasting, osteoporosis, and diabetes. Skeletal muscle wasting, which currently has no approved therapy, is a debilitating condition resulting from either reduced muscle protein synthesis or increased degradation. The imbalance in protein synthesis could occur from increased expression and function of muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle ring finger 1 (MuRF1), or decreased function of the IGF-I and phosphatidylinositol-3 kinase/Akt kinase pathways. We examined the effects of a nonsteroidal tissue selective androgen receptor modulator (SARM) and testosterone on glucocorticoid-induced muscle atrophy and castration-induced muscle atrophy. The SARM and testosterone propionate blocked the dexamethasone-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including Forkhead box O (FoxO). Dexamethasone caused a significant up-regulation in the expression of ubiquitin ligases, but testosterone propionate and SARM administration blocked this effect by phosphorylating FoxO. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of MAFbx and MuRF1 and down-regulation of IGF-I, all of which was attenuated by a SARM. The results suggest that levator ani atrophy caused by hypogonadism may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on up-regulation of MAFbx and MuRF1. Our studies provide the first evidence that glucocorticoid- and hypogonadism-induced muscle atrophy are mediated by distinct but overlapping mechanisms and that SARMs may provide a more effective and selective pharmacological approach to prevent glucocorticoid-induced muscle loss than steroidal androgen therapy.

Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR(+/-) mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR(+/-) mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR(+/-) mice to the same extent. Reduced muscle protein synthesis in mTOR(+/-) mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (Ser(307)) to the same extent in WT and mTOR(+/-) mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR(+/-) mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-I as well as tissue expression of TNFalpha, IL-6, or NOS2 did not differ between WT and mTOR(+/-) mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered mTOR(+/-) mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions.

Using transcriptomics to identify and validate novel biomarkers of human skeletal muscle cancer cachexia

BackgroundCancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. Skeletal muscle loss represents an established key feature yet there is no molecular understanding of the disease process. In fact, the postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment.MethodsRectus abdominis muscle biopsies were obtained from 65 upper gastrointestinal (UGI) cancer patients during open surgery and RNA profiling was performed on a subset of this cohort (n = 21) using the Affymetrix U133+2 platform. Quantitative analysis revealed a gene signature, which underwent technical validation and independent confirmation in a separate clinical cohort.ResultsQuantitative significance analysis of microarrays produced an 83-gene signature that was able to identify patients with greater than 5% weight loss, while this molecular profile was unrelated to markers of systemic inflammation. Selected genes correlating with weight loss were validated using quantitative real-time PCR and independently studied as general cachexia biomarkers in diaphragm and vastus lateralis from a second cohort (n = 13; UGI cancer patients). CaMKIIβ correlated positively with weight loss in all muscle groups and CaMKII protein levels were elevated in rectus abdominis. TIE1 was also positively associated with weight loss in both rectus abdominis and vastus lateralis muscle groups while other biomarkers demonstrated tissue-specific expression patterns. Candidates selected from the pre-clinical literature, including FOXO protein and ubiquitin E3 ligases, were not related to weight loss in this human clinical study. Furthermore, promoter analysis identified that the 83 weight loss-associated genes had fewer FOXO binding sites than expected by chance.ConclusionWe were able to discover and validate new molecular biomarkers of human cancer cachexia. The exercise activated genes CaMKIIβ and TIE1 related positively to weight-loss across muscle groups, indicating that this cachexia signature is not simply due to patient inactivity. Indeed, excessive CaMKIIβ activation is a potential mechanism for reduced muscle protein synthesis. Our genomics analysis also supports the view that the available preclinical models do not accurately reflect the molecular characteristics of human muscle from cancer cachexia patients.

The Effect of High Glucocorticoid Administration and Food Restriction on Rodent Skeletal Muscle Mitochondrial Function and Protein Metabolism

BackgroundGlucocorticoids levels are high in catabolic conditions but it is unclear how much of the catabolic effects are due to negative energy balance versus glucocorticoids and whether there are distinct effects on metabolism and functions of specific muscle proteins.Methodology/Principal FindingsWe determined whether 14 days of high dose methylprednisolone (MPred, 4 mg/kg/d) Vs food restriction (FR, food intake matched to MPred) in rats had different effects on muscle mitochondrial function and protein fractional synthesis rates (FSR). Lower weight loss (15%) occurred in FR than in MPred (30%) rats, while a 15% increase occurred saline-treated Controls. The per cent muscle loss was significantly greater for MPred than FR. Mitochondrial protein FSR in MPred rats was lower in soleus (51 and 43%, respectively) and plantaris (25 and 55%) than in FR, while similar decline in protein FSR of the mixed, sarcoplasmic, and myosin heavy chain occurred. Mitochondrial enzymatic activity and ATP production were unchanged in soleus while in plantaris cytochrome c oxidase activity was lower in FR than Control, and ATP production rate with pyruvate + malate in MPred plantaris was 28% lower in MPred. Branched-chain amino acid catabolic enzyme activities were higher in both FR and MPred rats indicating enhanced amino acid oxidation capacity.Conclusion/SignificanceMPred and FR had little impact on mitochondrial function but reduction in muscle protein synthesis occurred in MPred that could be explained on the basis of reduced food intake. A greater decline in proteolysis may explain lesser muscle loss in FR than in MPred rats.

The balancing act between the cellular processes of protein synthesis and breakdown: exercise as a model to understand the molecular mechanisms regulating muscle mass

the more we learn about skeletal muscle biology the more it becomes clear how important muscle tissue is for maintaining overall health. Specifically, muscle is important to provide sufficient strength/force production for locomotion and performing regular activities associated with daily living. In