The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb

Abstract

Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, Jefferson LS. The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb. Am J Physiol Endocrinol Metab 304: E229 -E236, 2013. First published November 27, 2012; doi:10.1152/ajpendo.00409.2012.-Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr 229 and Thr 389 in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr 229 by PDK1 is a prerequisite for phosphorylation of Thr 389 by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation.casting; anabolic resistance; 70-kilodalton ribosomal protein S6 kinase 1; 4E-binding protein 1; puromycin; mechanistic target of rapamycin complex 1; regulated in development and DNA damage responses 1/2 PROFOUND WASTING OF SKELETAL muscle, referred to as disuse atrophy, occurs in response to limb immobilization (10, 15, 16), limb suspension (40), and chronic bed rest (4,12,14). These conditions are associated with marked losses of skeletal muscle strength and function (21, 32), premature physical frailty (51), elevated health care costs, and an increased risk of mortality (36). Development of effective therapeutic strategies aimed at minimizing or preventing disuse atrophy will require a better understanding of the underlying molecular mechanisms that contribute to the loss of skeletal muscle mass. Maintenance of skeletal muscle mas...

Deletion of MLIP (Muscle-enriched A-type Lamin-interacting Protein) Leads to Cardiac Hyperactivation of Akt/Mammalian Target of Rapamycin (mTOR) and Impaired Cardiac Adaptation

“…To determine the rate of cardiac global protein synthesis, puromycin (Sigma; 40 mol⅐kg Ϫ1 of body weight) was intravenously injected to isofluorane-anesthetized animals (9). The heart was harvested 10 min after the injection.…”

“…5C), indicating activation of the mTOR pathway in these hearts. To investigate whether this alteration in mTOR signaling could result in changes in the protein synthesis process, we measured the rate of protein synthesis in vivo using the puromycin incorporation method (9). Despite the activation of mTOR pathway in Mlip Ϫ/Ϫ hearts, the steady state of protein synthesis was similar in Mlip Ϫ/Ϫ and Mlip ϩ/ϩ hearts (Fig.…”

From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy

“…Indeed, numerous studies demonstrated that a reduction in muscle protein fractional synthesis rate (FSR) occurs in the first days of immobilization [53,75,76]. This rapid reduction of protein synthesis persists for several weeks since a decrease of muscle FSR has been also reported in human skeletal muscle after 14 and 28 days of bed rest [77,78].…”

“…Interestingly, skeletal muscle exhibits alteration in PI3K/Akt/mTORC1 axis during immobilization [75,76,[80][81][82]. This phenomenon, called anabolic resistance, emphasizes the reduced response to anabolic stimuli [80,82].…”

Expression profile of plakin cross-linking proteins in short-term denervated mouse hindlimb skeletal muscle

“…[17][18][19] Atrophy can be induced experimentally through hindlimb suspension, hindlimb immobilization, chemical denervation (botulinum toxin), and surgical denervation (severing the nerve). [20][21][22][23][24] All these methods share the common trait of reducing or completely inhibiting neural input to the muscle, which induces atrophy. Denervation of adult muscles can revert the expression of specific genes to an embryonic expression profile.…”