Mechanical unloading induces muscle atrophy and bone loss; however, the time course and interdependence of these effects is not well defined. We subjected 4-month-old C57BL/6J mice to hindlimb suspension (HLS) for three weeks, sacrificing 12-16 mice on day (D) 0, 7, 14, and 21. Lean mass was 7-9% lower for HLS vs. control from D7-21. Absolute mass of the gastrocnemius (gastroc) decreased 8% by D7, and was maximally decreased 16% by D14 of HLS. mRNA levels of Atrogin-1 in the gastroc and quad were increased 99% and 122%, respectively, at D7 of HLS. Similar increases in MuRF1 mRNA levels occurred at D7. Both atrogenes returned to baseline by D14. Protein synthesis in gastroc and quad was reduced 30% from D7-14 of HLS, returning to baseline by D21. HLS decreased phosphorylation of SK61, a substrate of mammalian target of rapamycin (mTOR), on D7-21, while 4E-BP1 was not lower until D21. Cortical thickness of the femur and tibia did not decrease until D14 of HLS. Cortical bone of controls did not change over time. HLS mice had lower distal femur bone volume fraction (−22%) by D14; however, the effects of HLS were eliminated by D21 due to the decline of trabecular bone mass of controls. Femur strength was decreased approximately 13% by D14 of HLS, with no change in tibia mechanical properties at any time point. This investigation reveals that muscle atrophy precedes bone loss during unloading and may contribute to subsequent skeletal deficits. Countermeasures that preserve muscle may reduce bone loss induced by mechanical unloading or prolonged disuse. Trabecular bone loss with age, similar to that which occurs in mature astronauts, is superimposed on unloading. Preservation of muscle mass, cortical structure, and bone strength during the experiment suggests muscle may have a greater effect on cortical than trabecular bone.

BackgroundSkeletal muscle myopathy accompanying chronic alcohol misuse results in part from a decrease in protein synthesis typically observed in type II‐rich muscles that leads to muscle weakness. However, there is a paucity of studies investigating whether the alcohol‐induced weakness is intrinsic to the muscle or results primarily from the loss of muscle mass. The present study determines whether acute alcohol (ethanol) intoxication or chronic alcohol consumption decreases the intrinsic contractile function of muscle.MethodsAdult male mice were randomly assigned to the chronic alcohol group or given a binge dose of alcohol, and contractile characteristics of the extensor digitorum longus (EDL) were determined in vitro.ResultsThe weight and physiological cross‐sectional area (PCSA) of the EDL were decreased in alcohol‐fed mice. Maximum twitch and tetanic tension were also reduced, and there was a downward shift of the absolute force–frequency curve in alcohol‐fed mice. However, no alcohol‐induced changes were noted when these contractile parameters were normalized for the lower PCSA. Alcohol‐fed mice demonstrated greater fatigability, and alcohol‐induced decreases in postfatigue specific twitch and tetanic force were independent of a decreased PCSA. Furthermore, postfatigue recovery of muscle force over time was reduced. While alcohol did not alter the content of high‐energy phosphates or oxidative phosphorylation complexes I‐V, it did reduce myosin heavy chain and troponin‐T content. In contrast, contractile properties were not altered when examined 2 hours after binge alcohol.ConclusionsThese data demonstrate chronic alcohol consumption decreases isometric and tetanic tension development due to a reduction in muscle CSA, whereas the increased fatigability observed was independent of muscle mass. As none of the functional changes were produced by acute alcohol, which produced higher blood alcohol levels than chronic ingestion, our data suggest defects in intrinsic muscle contractility require sustained intake and appear independent of defects in basal energy production.

The etiology for the sepsis-induced leucine (Leu) resistance has not been fully elucidated and the present study investigated various aspects of amino acid activation of the mammalian target of rapamycin (mTOR). Sepsis in adult male rats decreased basal protein synthesis in gastrocnemius, associated with a reduction in mTOR activation as indicated by decreased 4EBP1 and S6K1 phosphorylation. The ability of oral Leu to increase protein synthesis and mTOR kinase after 1 h was largely prevented in sepsis. Sepsis increased CAT1, LAT2 and SNAT2 mRNA content 2- to 4-fold, but only the protein content for CAT1 (20% decrease) was significantly different. Conversely, sepsis decreased the proton-assisted amino acid transporter (PAT)-2 mRNA by 60%, but without a coordinate change in PAT2 protein. There was no sepsis or Leu effect on the protein content for RagA-D, LAMTOR-1 and -2, raptor, Rheb or mTOR in muscle. The binding of mTOR, PRAS40 and RagC to raptor did not differ for control and septic muscle in the basal condition; however, the Leu-induced decrease in PRAS40•raptor and increase in RagC•raptor seen in control muscle was absent in sepsis. The intracellular Leu concentration was increased in septic muscle, compared to basal control conditions, and oral Leu further increased the intracellular Leu concentration similarly in both control and septic rats. Hence, while alterations in select amino acid transporters are not associated with development of sepsis-induced Leu-resistance, the Leu-stimulated binding of raptor with RagC and the recruitment of mTOR/raptor to the endosome-lysosomal compartment may partially explain the inability of Leu to fully active mTOR and muscle protein synthesis.

Small molecule nonpeptidyl molecules are potentially attractive drug candidates as adjunct therapies in the treatment of sepsis-induced metabolic complications. As such, the current study investigates the use of aurintricarboxylic acid (ATA), which stimulates insulin-like growth factor (IGF)-I receptor and AKT signaling, for its ability to ameliorate the protein metabolic effects of endotoxin (LPS) + interferon (IFN)γ in C2C12 myotubes and sepsis in skeletal muscle. ATA dose- and time-dependently increases mTOR-dependent protein synthesis. Pretreatment with ATA prevents the LPS/IFNγ-induced decrease in protein synthesis at least in part by maintaining mTOR kinase activity, while post-treatment with ATA is able to increase protein synthesis when added up to 6 h after LPS/IFNγ. ATA also reverses the amino acid resistance which is detected in response to nutrient deprivation. Conversely, ATA decreases the basal rate of protein degradation and prevents the LPS/IFNγ-increase in proteolysis, and the latter change is associated reduced atrogin-1 and MuRF1 mRNA. The ability of ATA to antagonize LPS/IFNγ-induced changes in protein metabolism were associated with its ability to prevent the increases in IL-6 and NOS2 and decreases in IGF-I. In vivo studies indicate ATA acutely increases skeletal muscle, but not cardiac, protein synthesis, and attenuates the loss of lean body mass over 5 days. These data suggest ATA and other small molecule agonists of endogenous anabolic hormones may prove beneficial in treating sepsis by decreasing the inflammatory response and improving muscle protein balance.