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Regulation of Hepatic Glucose Metabolism. SEUNG-HOI KOO Sungkyunkwan University, School of Medicine 05-30-2008. Insulin regulates glucose homeostasis. Annu Rev Physiol. 2006;68:123-58. Insulin regulates Foxo activity. Foxo1 regulates hepatic glucose production. - PowerPoint PPT Presentation
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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 CHANDAGNF