Insulin and Amino Acid Signaling Through mTOR in Perfused Rat Liver Jessica Biegler

Objective Define how amino acids interact with insulin to enhance signaling through mTOR to regulatory mechanisms that control mRNA translation in perfused rat liver.

Experimental Design Male, Sprague-Dawley rats g In situ liver perfusion –20 minute perfusion –Amino acids: 1X or 4X the concentrations found in arterial plasma of a postabsorptive rat –Insulin: 0-, 1-, or 10- nM final concentration –Control: saline perfusion

Traditional Signaling TSC1 TSC2 insulin PI3-kinase Akt Rheb G  L. raptor. mTOR amino acids ? ? ? p70 S6K1 rpS6 4E-BP1 eIF4E mRNA Translation hVps34 IRS-1

Neither Insulin Nor Amino Acids Stimulate a Significant Phosphorylation of IRS-1 on 636/639 P Value Interaction Insulin Amino Acid N=3

Insulin Stimulates AKT S473 Phosphorylation Independently of Amino Acids P Value Interaction Insulin< * Amino Acid N=6

Neither Insulin Nor Amino Acids Stimulate a Significant Association of TSC1/2 P Value Interaction Insulin Amino Acid N=9

Insulin Stimulates PRAS Phosphorylation Independently of Amino Acids P Value Interaction Insulin * Amino Acid N=6

Neither Insulin Nor Amino Acids Stimulate a Significant Phosphorylation of TSC1/2 on 1462 P Value Interaction Insulin Amino Acid N= 9

Neither Insulin Nor Amino Acids Stimulate a Significant Effect on REDD 1 P Value Interaction Insulin Amino Acid N=6

Neither Insulin Nor Amino Acids Stimulate a Significant Effect on FKBP 38 P Value Interaction Insulin Amino Acid N=6

Neither Insulin Nor Amino Acids Stimulate a Significant Effect on ATF 4 P Value Interaction Insulin Amino Acid N=6

Insulin and Amino Acids Stimulate eIF2α Phosphorylation P Value Interaction Insulin * Amino Acid * N=6

Insulin and Amino Acids Stimulate a Significant Phosphorylation of mTOR on 2448 P Value Interaction Insulin * Amino Acid * N=9

Insulin Nor Amino Acids Stimulate a Significant Association of VPS34 with mTOR P Value Interaction Insulin Amino Acid N=3

Amino Acids Act Synergistically with Insulin to Enhance Translation Initiation P Value Interaction * Insulin * Amino Acid * N=9

Amino Acids Act Synergistically with Insulin in the Phosphorylation of eIF4G on 1108 P Value Interaction Insulin * Amino Acid< * N=6

Amino Acids Act Synergistically with Insulin to Enhance p70 S6K1 Phosphorylation P Value Interaction * Insulin< * Amino Acid< * N=9

Amino Acids Act Synergistically with Insulin to Enhance p70 S6K1 Phosphorylation on 389 P Value Interaction * Insulin * Amino Acid< * N=3

Amino Acids are Required for Insulin-Stimulated Protein Synthesis P Value Interaction * Insulin Amino Acid< * N=9

Proposed Signaling mTORC2 Insulin ? IRS 1 PI3K PIP2PIP3 PTEN AKT PDK1 Amino Acids eIF2α ? ? ATF 4 REDD 1 TSC1 TSC2 PRAS 40 ? ? mTORC1 Rheb ? ?? ?? ? ? ? PLD FKBP 38 RAG B RAG C VPS 34 ? ? 4E-BP1 eIF4E eIF4G S6K 1 S6

Conclusions Amino acids are necessary for insulin- induced signaling through mTOR in perfused rat liver. Amino acids signaling may be occuring through the association of various proteins with mTOR.

Overall Summary Insulin and amino acids act synergistically to stimulate signaling through mTOR in perfused rat liver. –Vps34/mTOR interaction