The hypothesis of the present study was that rats subjected to shortterm unilateral hindlimb immobilization would incur skeletal muscle wasting and concomitant alterations in protein synthesis, controllers of translation, and indexes of protein degradation. Rats were unilaterally casted for 1, 3, or 5 days to avoid complications associated with other disuse models. In the casted limb, gastrocnemius wet weight decreased 12% after 3 days and thereafter remained constant. In contrast, the contralateral control leg displayed a steady growth rate over time. The rate of protein synthesis and translational efficiency were unchanged in the immobilized muscle at day 5. The total amount and phosphorylation state of regulators of translational initiation and elongation were unaltered. The mRNA contents of polyubiquitin and the ubiquitin ligases muscle atrophy F-box (MAFbx)/Atrogin-1 and muscle RING finger 1 (MuRF1) were elevated in immobilized muscle at all time points, with peak expression occurring at day 3. Daily injection of the type II glucocorticoid receptor antagonist RU-486 did not prevent decreases in gastrocnemius wet weight nor increases in mRNA for MAFbx/Atrogin-1 and MuRF1. However, in vivo administration of the proteasome inhibitor Velcade prevented 53% of wet weight loss associated with 3 days of immobilization. These data suggest that the loss of skeletal muscle mass in this model of disuse appears to be glucocorticoid independent, can be partially rescued with a potent proteasome inhibitor, and is associated with enhanced mRNA expression of multiple factors that contribute to ubiquitinproteasome-dependent degradation and are likely to control the remodeling of immobilized skeletal muscle during atrophy.Atrogin-1; muscle RING finger 1; disuse; eukaryotic initiation factor; translation SKELETAL MUSCLE DEMONSTRATES a remarkable degree of plasticity in response to alterations in mechanical loading. Removal of normal weight-bearing stress results in quantitative and qualitative adaptations in protein content that are phenotypically epitomized by decreased muscle wet weight, fiber diameter, force output, fatigue resistance, and a slow-to-fast

hypothesis of the present study was that exposure of differentiated muscle cells to agonists of the AMP-activated protein kinase (AMPK) would increase the mRNA content of the muscle-specific ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). C 2C12 cells were incubated with incremental doses of 5-aminoimidazol-4-carboximide ribonucleoside (AICAR) or metformin for 24 h. Both MAFbx and MuRF1 mRNA increased dose dependently in response to these AMPK activators. AICAR, metformin, and 2-deoxy-D-glucose produced time-dependent alterations in ubiquitin ligase expression, typified by a biphasic pattern of expression marked by an acute repression followed by a sustained induction. AMPK-activating treatments in conjunction with dexamethasone produced a pronounced synergistic effect on ligase mRNA expression at later time points. This cooperative response occurred in the absence of a dexamethasone-dependent increase in AMPK expression or activity, as determined by immunoblotting for phosphorylation and expression of AMPK␣ and its downstream target acetyl-CoA carboxylase (ACC). These responses elicited by AMPK activation singly or in combination with dexamethasone did not extend to the mRNA expression of the UBR box family E3s UBR1/E3␣I and UBR2/E3␣II. Treatment with the AMPK inhibitor compound C prevented increases in MAFbx and MuRF1 mRNA in response to serum deprivation, as well as AICAR and dexamethasone treatment individually or jointly. Stimulation of AMPK activity in vivo via AICAR injection increased both MAFbx and MuRF1 mRNA in murine skeletal muscle. These data suggest that activation of AMPK in skeletal muscle results in a specific upregulation of MAFbx and MuRF1, responses that are reminiscent of the proposed atrophic transcriptional program executed under various conditions of skeletal muscle wasting. Therefore, AMPK may be a critical component of the intercalated network of signaling pathways governing skeletal muscle atrophy, where its input acts to modify anti-and proatrophic signals to influence gene expression in reaction to catabolic perturbations.atrogin-1; proteolysis; 2-deoxyglucose FOR PROPER GROWTH AND SURVIVAL, all organisms require means of responding to diverse inputs originating from a discontinuous environmental milieu. Failure to both properly integrate these signals and accordingly adjust metabolism within favorable physiological limits may ultimately result in maladaptation, disease, and death. One critical component of this control system in mammalian systems is the AMP-activated protein kinase (AMPK). AMPK functions as a heterotrimer (consisting of a catalytic ␣-subunit and regulatory ␤-and ␥-subunits), which senses alterations in the AMP-to-ATP ratio within cells (15,25,35). In response to energy-depleting stressors (which consequently increase cellular AMP concentrations), AMPK acts to balance energy consumption with production by suppressing ATP-expensive processes and activating ATP-repleting ones. Regulation of its activity is achieved in an elegant fashion...

The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-α (TNF-α) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-α to cultured C2C12 myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-α, and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions.

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