1. SEUNG-HOI KOO Sungkyunkwan University, School of Medicine 05-30-2008 Regulation of Hepatic Glucose Metabolism

2. Insulin regulates glucose homeostasis Annu Rev Physiol. 2006;68:123-58

3. Insulin regulates Foxo activity

4. Foxo1 regulates hepatic glucose production Transcriptionally activate gluconeogenic genes during fasting. Highly active in livers of diabetic mice due to the hypophosphorylaton of 3 major Akt sites. Attractive target for controlling hepatic glucose production.

5. Arden et al. Arch Biochem Biophys. 2002 Insulin regulates Foxo activity

6. FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384- well containing plasmid DNA from 4800 MGC collection High-throughput transfection & imaging

7. FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384- well containing plasmid DNA from 4800 MGC collection Addition of U2OS cells to each well High-throughput transfection & imaging

8. FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384- well containing plasmid DNA from 4800 MGC collection Addition of U2OS cells to each well Fixation, nuclei-staining (DAPI), and imaging on inverted fluorescence microscope High-throughput transfection & imaging

9. FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384- well containing plasmid DNA from 4800 MGC collection Addition of U2OS cells to each well Fixation, nuclei-staining (DAPI), and imaging on inverted fluorescence microscope Fractional fluorescence in the nucleus (FLIN value) determined for all GFP-positive cells High-throughput transfection & imaging

10. FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384- well containing plasmid DNA from 4800 MGC collection Addition of U2OS cells to each well Fixation, nuclei-staining (DAPI), and imaging on inverted fluorescence microscope Fractional fluorescence in the nucleus (FLIN value) determined for all GFP-positive cells Percentages of cells with cytoplasmic, mixed, and nuclear GFP-FOXO1 reported (GFP-FOXO1 fluorescence > 2-fold over background). High-throughput transfection & imaging

11. Screen for modulators of FOXO1 subcellular localization

13. Alonso et al Cell. 2004 Family of Protein Tyrosine Phosphatases

14. PTPs are a diverse enzyme family with at least 107 members in the human genome PTP-1b : inhibitors of insulin signaling - PTP-1b KO mice exhibit improved insulin sensitivity compared to wild-type controls and are resistant to weight gain when fed a high-fat diet - PTP-1b is localized to the cytoplasmic surface of the endoplasmic reticulum, and dephosphorylates receptor tyrosine kinases after receptor endocytosis Family of Protein Tyrosine Phosphatases

15. Possesses a lipid binding domain homologous to Sec14p, a yeast protein with phosphatidylinositol (PtdIns) transferase activity. Regulates the secretory pathway through dephosphorylation of the fusion protein N- ethylmaleimide-sensitive factor Regulation of intracellular traffic of the secretory pathway in T cells. PTP-MEG2

16. PTP-MEG2 modulates insulin signaling in cultured cells HepG2

17. PTP-MEG2 modulates insulin signaling in cultured cells HepG2 Primary Hepatocytes

18. PTP-MEG2 modulates insulin signaling in cultured cells

20. PTP-MEG2 inhibits suppression of hepatic glucose output

22. Knockdown of PTP-MEG2 potentiates insulin activity in hepatocytes

24. Knockdown of PTP-MEG2 in diabetic mice results in insulin sensitization

28. Conclusion PTP-MEG2 expression blunts insulin-mediated transcriptional repression of gluconeogenic genes.

29. Conclusion PTP-MEG2 expression blunts insulin-mediated transcriptional repression of gluconeogenic genes. PTP-MEG2 regulates insulin signal transduction by attenuating the activation of the insulin receptor.

30. Conclusion PTP-MEG2 expression blunts insulin-mediated transcriptional repression of gluconeogenic genes. PTP-MEG2 regulates insulin signal transduction by attenuating the activation of the insulin receptor. Reduction of PTP-MEG2 expression levels with Ad RNAi in the livers of diabetic (db/db) mice resulted in a reversal of insulin resistance and hyperglycemia.

31. Conclusion PTP-MEG2 expression blunts insulin-mediated transcriptional repression of gluconeogenic genes. PTP-MEG2 regulates insulin signal transduction by attenuating the activation of the insulin receptor. Reduction of PTP-MEG2 expression levels with Ad RNAi in the livers of diabetic (db/db) mice resulted in a reversal of insulin resistance and hyperglycemia. These results indicate that PTP-MEG2 regulates glucose homeostasis and hepatic insulin action through the modulation of insulin receptor signaling.

32. Regulation of insulin signaling pathways

33. Acknowledgment Salk Inst MARC MONTMINY Susan Hedrick Sungkyunkwan U SEUNG-HOI KOO Young-Sil Yoon Dongryeol Ryu Hee-Yeon Jo Woo-Young Seo Kyeoung Jin Oh Min-Woo Lee Scripps Inst PETER SHULTZ Charles Cho SUMIT CHANDA GNF