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

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

3. Experimental Design Male, Sprague-Dawley rats 125-150 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

4. 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

5. Neither Insulin Nor Amino Acids Stimulate a Significant Phosphorylation of IRS-1 on 636/639 P Value Interaction0.9268 Insulin0.5523 Amino Acid0.1571 N=3

6. Insulin Stimulates AKT S473 Phosphorylation Independently of Amino Acids P Value Interaction0.6544 Insulin<0.0001 * Amino Acid0.5584 N=6

7. Neither Insulin Nor Amino Acids Stimulate a Significant Association of TSC1/2 P Value Interaction0.4315 Insulin0.8317 Amino Acid0.2412 N=9

8. Insulin Stimulates PRAS40 246 Phosphorylation Independently of Amino Acids P Value Interaction0.4593 Insulin0.0036 * Amino Acid0.2613 N=6

9. Neither Insulin Nor Amino Acids Stimulate a Significant Phosphorylation of TSC1/2 on 1462 P Value Interaction0.4905 Insulin0.1901 Amino Acid0.4702 N= 9

10. Neither Insulin Nor Amino Acids Stimulate a Significant Effect on REDD 1 P Value Interaction0.3113 Insulin0.9258 Amino Acid0.6184 N=6

11. Neither Insulin Nor Amino Acids Stimulate a Significant Effect on FKBP 38 P Value Interaction0.4483 Insulin0.5751 Amino Acid0.9259 N=6

12. Neither Insulin Nor Amino Acids Stimulate a Significant Effect on ATF 4 P Value Interaction0.1694 Insulin0.8545 Amino Acid0.1164 N=6

13. Insulin and Amino Acids Stimulate eIF2α Phosphorylation P Value Interaction0.2318 Insulin0.0228 * Amino Acid0.0328 * N=6

14. Insulin and Amino Acids Stimulate a Significant Phosphorylation of mTOR on 2448 P Value Interaction0.8195 Insulin0.0476 * Amino Acid0.0110 * N=9

15. Insulin Nor Amino Acids Stimulate a Significant Association of VPS34 with mTOR P Value Interaction0.1872 Insulin0.8049 Amino Acid0.1164 N=3

16. Amino Acids Act Synergistically with Insulin to Enhance Translation Initiation P Value Interaction0.0416 * Insulin0.0218 * Amino Acid0.0002 * N=9

17. Amino Acids Act Synergistically with Insulin in the Phosphorylation of eIF4G on 1108 P Value Interaction0.1128 Insulin0.0334 * Amino Acid<0.0001 * N=6

18. Amino Acids Act Synergistically with Insulin to Enhance p70 S6K1 Phosphorylation P Value Interaction0.0065 * Insulin<0.0001 * Amino Acid<0.0001 * N=9

19. Amino Acids Act Synergistically with Insulin to Enhance p70 S6K1 Phosphorylation on 389 P Value Interaction0.0059 * Insulin0.0011 * Amino Acid<0.0001 * N=3

20. Amino Acids are Required for Insulin-Stimulated Protein Synthesis P Value Interaction0.0019 * Insulin0.0524 Amino Acid<0.0001 * N=9

21. 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

22. 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.

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