Regulation of Hepatic Glucose Metabolism

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

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

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

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

8. High-throughput transfection & imaging • 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

9. High-throughput transfection & imaging • 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

10. High-throughput transfection & imaging • 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).

11. Screen for modulators of FOXO1 subcellular localization

12. Screen for modulators of FOXO1 subcellular localization

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

14. Family of Protein Tyrosine Phosphatases • 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

15. PTP-MEG2 • 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.

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

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

18. PTP-MEG2 modulates insulin signaling in cultured cells

19. PTP-MEG2 modulates insulin signaling in cultured cells

20. PTP-MEG2 inhibits suppression of hepatic glucose output

21. PTP-MEG2 inhibits suppression of hepatic glucose output

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

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

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

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

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

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