The overall objective is to develop a mechanistic approach and improved understanding of potential molecular targets in the management of disordered protein turnover and muscle atrophy in the diaphragm of a clinically relevant animal model of severe short-term nutritional deprivation (ND). Hypothesis 1: Down-regulation of the PI3K/Akt/mTOR signal transduction pathway is an important determinant of disordered synthesis of muscle proteins and atrophy with ND. Reduced IGF-1 levels are a major signal responsible for this down-regulation. Stimulation of PI3K/Akt/mTOR signal transduction pathway by administration of IGF-1 will augment the synthesis of muscle proteins by enhancing signal transduction pathways mediating translation initiation with attenuation of muscle atrophy.
Aim 1 : We propose to measure in a time series (i.e., days 1 & 4) in severe ND, the phosphorylation of Akt and Ser2448,an important regulatory domain in mTOR, together with downstream targets important in translation initiation of muscle proteins (i.e., phosphorylation states of 4E-BPI and p70S6K) and the phosphorylation of ERK1/2 and GSK3, 2 other pathways through which IGF-1 could act. The above will be measured with and without the infusion of IGF-1 and correlated with 1) muscle protein synthesis and degradation including an analysis of the turnover of specific protein pools (MHC, sarcoplasmic, mitochondrial); 2) diaphragm mass and fiber size; and 3) diaphragm specific force. Hypothesis 2: The impact of rapamycin, an inhibitor of mTOR, on IGF-1 rescue in the ND model is unknown. Possibilities include marked attenuation of the beneficial effect of IGF-1 or maintained rescue due to activation of alternate pathways.
Aim 2 : We propose to administer rapamycin to ND animals infused with IGF-I and to measure the key proteins mediating IGF-1 signal transduction pathways together with outcome measures as outlined in aim #1. Hypothesis 3: Enhanced muscle protein degradation with severe ND is mediated in large part by activation of the ubiquitin-proteasome pathway. While IGF-1 infusion may potentially attenuate muscle protein breakdown with ND, the addition of an inhibitor of the ubiquitin proteasome mediated proteolysis (eicosapentaenoic acid; EPA) to IGF-1 infusion will further attenuate or reverse muscle fiber atrophy. The provision of EPA alone to ND animals will determine the relative importance of muscle proteolysis to disordered turnover in this model.
Aim 3 : We propose to administer EPA with and without IGF-1 infusion to ND animals. We will evaluate gene and protein expression of key components of the ubiquitin-proteasome pathway. These will include: the newly described muscle specific E3 ligases (MuRF1 and MAFbx), key enzymes mediating the N-end rule pathway (E2-I4k and E3alpha ), polyubiquitin, proteasome subunits (C2 & C8) and ubiquitin conjugated protein content. The above will be correlated with outcome measures as outlined in aim #1. Hypothesis 4: Increased endogenous corticosteroid (CS) production is present with severe ND, which contributes to muscle fiber atrophy by suppressing muscle protein synthesis and enhancing degradation through the ubiquitin-proteasome pathways. Suppression of CS effects by administration of a CS receptor blocker (RU38486) will reverse CS induced effects on muscle protein turnover and enable one to evaluate the contribution of endogenous CS to fiber atrophy.
Aim 4 : We propose to administer RU38486 to animals subjected to severe short-term ND. We will evaluate gene and protein expression of key components of the ubiquitin-proteasome pathway, as outlined in aim #3, as well as markers of signal transduction and outcome measures as outlines in aim #1.
