The principal hypothesis of the proposed studies are that physiologic variations and amino acid supply, acting in concert with insulin, alter the synthesis of both total and specific hepatic routines via coordinated modulation of the eIF2/eIF2B, eIF4F and P70 S 6 K regulatory steps in translation initiation and the RNA and polymerase 1 step in ribosome bio synthesis. The investigators proposed tested this hypothesis by addressing five games. The first is to assess the contributions and sites of action of amino acids supply and insulin in modulated hepatic protein synthesis under various nutritional states. The second is to identify the mechanisms by which amino acids supply and insulin act to modulate eIF2/eIF2B, eIF4F and P70 S 6 K regulatory steps in translation initiation. The third is to correlate variations in ribosome synthesis with S6 phosphorylation and preferential translation of ribosomal protein mRNAs, the fourth is to identify the mechanism by which amino acid supply and insulin act to modulate transcription of ribosomal RNA genes and finally to identify the relevant signaling pathways through which amino acids and insulin act to modulate the eIF2/eIF2B, eIF4F and P70 S 6 K regulatory steps in translation initiation and RNA polymerase 1 step in ribosome biogenesis. The experimental plan for testing the overall hypothesis employs both in vivo and in vitro models for generating variations in amino acid supply and insulin availability, analyzing activity, and phosphorylation state of various translation initiation and transcription factors and their regulatory proteins. The PI will employ dominant interfering variants and gene disruption to validate the role of specific proteins, assess the role rapamycin-sensitive and insensitive signaling pathways. Overall, the studies described in this proposal should provide new insights into the biochemical and molecular mechanisms involved in the regulation of protein synthesis by liver.
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