SEUNG-HOI KOO

Sungkyunkwan University, School of Medicine05-30-2008

Regulation of Hepatic Glucose Metabolism

Insulin regulates glucose homeostasis

Annu Rev Physiol. 2006;68:123-58

Insulin regulates Foxo activity

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.

Arden et al. Arch Biochem Biophys. 2002

Insulin regulates Foxo activity

• FuGENE 6 + pcDNA-GFP-FOXO1 onto prespotted 384-well containing plasmid DNA from 4800 MGC collection

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

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

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

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).

High-throughput transfection & imaging

Screen for modulators of FOXO1 subcellular localization

Screen for modulators of FOXO1 subcellular localization

Alonso et al Cell. 2004

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

Family of Protein Tyrosine Phosphatases

• 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

PTP-MEG2 modulates insulin signaling in cultured cells

HepG2

PTP-MEG2 modulates insulin signaling in cultured cells

HepG2 Primary Hepatocytes

PTP-MEG2 modulates insulin signaling in cultured cells

PTP-MEG2 modulates insulin signaling in cultured cells

PTP-MEG2 inhibits suppression of hepatic glucose output

PTP-MEG2 inhibits suppression of hepatic glucose output

Knockdown of PTP-MEG2 potentiates insulin activity in hepatocytes

Knockdown of PTP-MEG2 potentiates insulin activity in hepatocytes

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

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

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

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

Conclusion

• PTP-MEG2 expression blunts insulin-mediated

transcriptional repression of gluconeogenic genes.

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.

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.

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.

Regulation of insulin signaling pathways

Acknowledgment

Salk InstMARC MONTMINYSusan Hedrick

Sungkyunkwan USEUNG-HOI KOOYoung-Sil YoonDongryeol RyuHee-Yeon JoWoo-Young SeoKyeoung Jin OhMin-Woo Lee

Scripps InstPETER SHULTZCharles Cho

SUMIT CHANDA

GNF