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Insulin Signalling BCH 2021, November 2006 Amira Klip The Hospital for Sick Children, Toronto

Insulin Signalling BCH 2021, November 2006 Amira Klip The Hospital for Sick Children, Toronto Amira@sickkids.ca. Our brain is the main glucose consumer…between meals. Blood glucose. Liver (glucose store). brain. Fat cells. pancreas. All other organs. Skeletal muscle.

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Insulin Signalling BCH 2021, November 2006 Amira Klip The Hospital for Sick Children, Toronto

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  1. Insulin Signalling BCH 2021, November 2006 Amira Klip The Hospital for Sick Children, Toronto Amira@sickkids.ca

  2. Our brain is the main glucose consumer…between meals Blood glucose Liver (glucose store) brain Fat cells pancreas All other organs Skeletal muscle

  3. Dietary glucose is stored in the liver, muscle and fat cells Blood glucose liver brain Fat cells pancreas insulin Skeletal muscle

  4. Pancreas, liver, fat, muscle and brain determine glucose homeostasis Blood glucose insulin Adapted from Nature 414, 2001 Skeletal muscle

  5. Insulin directly acts on liver, muscle and fat Liver Adipose tissue Muscle

  6. The insulin receptor dimer From Chang et al (2004) Mol Med 10:65

  7. Insulin receptor conformation From Yip and Ottensmeyer (2003) JBC 278: 27329

  8. Insulin and Insulin-like Receptors

  9. Structure relationships of IRS family IRS2: human Irs2: mouse DroIRS: drosophila (chico)

  10. Levels of insulin action Acute: glucose and lipid metabolism Intermediate: Proteins synthesis Long-acting: Gene expression

  11. Insulin receptor: constitutive dimer IRS-1,2,3,4 PM NPXY PH PTB IIIIIIII adaptor PHIP (pY) (pY)n Grb GEF shc Sos Small G Ras Raf Ser/thr kinases MEK Tyr/thr kinase ERK P90 rsk gene expression

  12. Insulin receptor PM IRS-1,2,3,4 p85 shc (pY) Grb PI kinase Sos p110 Ras Ser/thr kinases PDK1,2 PKB/ Akt1,2,3 - Raf Gsk-3 mTOR MEK - ERK eIF-2B eIF-4E 4E-BP1 + - P90 rsk + mRNA translation and protein synthesis

  13. Regulation of metabolism by insulin

  14. Insulin stimulates glucose uptake into muscle and fat tissues, via GLUT4 GLUT4 is the major insulin-responsive glucose transporter Pancreas

  15. Glucose Transporter (GLUT) Family (SLC2)

  16. Glucose enters muscles through GLUT4 PM PM cytosol cytosol

  17. GLUT4 continuously recycles, insulin increases GLUT4 at the surface exocytosis What are the signals involved?

  18. Insulin receptor GLUT4 trans-location PM IRS-1,2 p85 AS160 is a Rab-GAP shc (pY) Grb PI kinase Sos p110 Ras Ser/thr kinases PDK1,2 PKB/Akt1,2,3 AS160 Raf MEK mTOR eIF-4E ERK eIF-2B + 4E-BP1 + P90 rsk -

  19. Signal transduction versatility, ups and downs: • isoforms • cellular location • intensity (amplitude) • duration • frequency (transient or sustained) • feedback regulation ( + or - )

  20. NR IRS-1 IRS-2 NR IRS-1 IRS-2 siRNA: siRNA: IRS-1 IRS-2 120 120 100 100 IRS-1 expression level 80 80 IRS-2 expression level 60 60 ** 40 40 *** 20 20 0 0 NR IRS-1 IRS-2 siRNA: NR IRS-1 IRS-2 siRNA: siRNA-mediated knock-down of IRS-1 or IRS-2 Huang, Thirone et al (2005) J Biol Chem 280: 1226

  21. IRS-1, but not IRS-2, is required for insulin-induced GLUT4 translocation siRNA to IRS-1 siRNA to IRS-2 *** 100 100 80 80 ** % of maximal response 60 % of maximal response 60 40 40 20 20 0 0 Insulin (nM) Insulin, nM 5 100 100 5

  22. IRS-1, but not IRS-2, is required for insulin-stimulation of glucose uptake Glucose uptake siRNA: IRS-1 siRNA: IRS-2 ** 100 100 80 80 ** 60 60 % of maximal response % of maximal response 40 40 20 20 0 0 Insulin (nM) Insulin (nM) 100 5 100 5

  23. IRS-1 and IRS-2 have complementary but not redundant functions in muscle cells IRS-1 IRS-2 PI 3-kinase PI 3- kinase p38 ERK Akt1 Akt2 actin Mitogenic pathway Lipid metab, liver & muscle GLUT4 translocation Glc metab, liver Huang, Thirone et al (2005) J Biol Chem 280: 1226

  24. Insulin signalling:an integrated circuit Thong et al, (2005) Physiology 20:271

  25. The circuit has built-in feedback inhibition P P Pser Pser IRS-1 Pser Pser Rho Kinase Pser pY pY PI 3-kinase p85 p110 Grb2 SOS Ras Raf MEK ERKs MKKK MKK JNK AMPK PDKs mTOR S6Kinase PKCl/z Akt/PKB GSK3 IL-1 R mPLK

  26. PKCz IKK PKC JNK mTOR Ser 318, 570 Ser 307 Ser 408 IRS-1 Ser 302 Ser 612 mTOR ERK PI3K Ser 24 Ser 632 Ser 9 Ser 789 mPLK Akt Rho kinase pY pY PI 3-kinase p85 p110 GSK3 AMPK All roads lead to IRS-1

  27. Additional negative regulation

  28. Insulin PI 3-kinase Akt phosphorylation AS160 Rab-GTP active Rab-GDP inactive GLUT4 translocation GEF ? Insulin AS-160: a Rab-GAP S588 S341 T642 S318 S751 S570 R973 1 119 189 357 439 917 1136 1299 ‘4P’ mutant S318A, S588A, S751A, T642A cannot be phosphorylated: Rab-GDP prevails GAP domain PTB PTB As per Lienhard et al

  29. Insulin-induced GLUT4-vesicle translocation and fusion in rounded-up myoblasts N Intact cells Basal Insulin surface Myc Permeabilized cells Myc C-terminus

  30. AS160 mutant 4P prevents the insulin and PI-3,4,5-P3-induced GLUT4 translocation 3.0 untransfected 2.5 4P 2.0 Cell surface GLUT4myc (fold over basal untransfected) 1.5 1.0 0.5 0.0 Basal 100 nM Insulin 10 M PIP3+Carrier Hypertonic Sucrose Thong et al, unpublished

  31. AS160-4P allows significant GLUT4 arrival, but eliminates GLUT4 fusion Varinder Randhawa, Alex Cheng

  32. Insulin signals leading to GLUT4 p85 p110 PKCl/z Akt AS160 fusion Insulin Receptor P P IRS-1 P P P P PI 3-Kinase PDK P P P P GLUT4 translocation

  33. Recurrent themes in insulin signalling Insulin Receptor Insulin Receptor P P P P IRS-1 IRS-1 PI 3-Kinase PI 3-Kinase P P P P P P P P PDK PDK AMPK P P P P p85 p85 p110 p110 TSC1,2 (GAP) AS160 (GAP) PKC the ‘hand brakes’ Stress kinases Rab 8,10,14? Rheb mTOR Akt Akt GLUT4 translocation and glucose uptake, Glycogen synthesis Protein synthesis ATP expender ATP provider

  34. IR CAP Cbl TC10 Is that all it takes? If not complicated enough… a new signalling pathway involving APS-Cap-Cbl-C3G-TC10 was proposed (Saltiel and Pessin) Actin dynamics and GLUT4 translocation

  35. A second signalling pathway? IR CAP Cbl TC10 Rac Actin dynamics Exocyst Insulin Receptor P P IRS-1 P P P P p85 p110 PI 3,4,5-P3 PDK Akt AS160 GLUT4 translocation

  36. CAP-Cbl-TC10 do not participate in actin remodelling in myoblasts: IR CAP Cbl TC10 • CAP is expressed only in myotubes (not myoblasts) • Cbl is phosphorylated in response to insulin only in myotubes • Endogenous TC10 is not detectable with available antibodies • Transfected TC10 is GTP-loaded in myoblasts and myotubes • stimulated with insulin • TC10 mutants do not prevent insulin-dependent actin • remodelling into a mesh, nor GLUT4 translocation

  37. Insulin signalling pathway Actin dynamics Insulin Receptor P P IRS-1 P P P P p85 p110 PI 3,4,5-P3 PDK Akt AS160 GLUT4 translocation

  38. Insulin-dependent actin filament remodelling Basal Insulin Myotubes Myoblasts Rounded-up myoblasts Acta Physiol Scand 178: 297-308 (2003)

  39. Disrupting actin polymerization prevents insulin-dependent GLUT4myc externalization * Hypertonicity 2.5 Basal • Insulin effect also prevented by: • Jasplakinolide • Swinholide A Insulin 2 1.5 Cell surface GLUT4myc 1 0.5 0 Control Cyto D Latrunculin B Adapted from J. Clin. Invest. 108: 371-381 (2001) And Mol. Biol. Cell,

  40. Insulin signalling pathway ? Actin dynamics Insulin Receptor P P IRS-1 P P P P p85 p110 PI 3,4,5-P3 PDK Akt AS160 GLUT4 translocation

  41. General Biological Roles Cell cycle regulation Vesicle traffic Morphogenesis Cell migration Actin remodelling GTPases in insulin signalling RhoA (fibroblasts) Cdc 42 (adipocytes) TC10 (adipocytes) Rac (muscle) Activation signal GTP GDP GEF GDP GTP Rho Rho Inactive Active GAP Pi Rho GTPases TC10 Takai et al 2002

  42. GTPase GTP Rac activation in response to insulin Serum depletion Stimulation (100 nM Insulin) & lysis hPAK1B – CRIB aa56-272: + GST Lellean JeBailey

  43. 3.5 Basal Insulin 3.0 2.5 2.0 1.5 1.0 0.5 0 UT WT-Rac DN-Rac DN Rac inhibits insulin-induced actin remodelling and GLUT4 translocation DN-Rac Actin Basal Fold GLUT4myc Translocation Insulin Lellean JeBailey

  44. Rac1 knockdown Reduces GLUT4 translocation Basal Insulin Insulin PI3K RAC p <0.001 GTP Actin Remodelling/ GLUT4 Translocation siUR siRac 1 2.5 2.0 1.5 Fold GLUT4 Translocation 1.0 0.5 0 Adapted from JeBailey et al (2006) Diabetes in press

  45. siRac 1 Inhibits PAK, LIMK but not Akt Activation Insulin PI3K RAC GTP PAK P LIMK P Akt P Insulin - + - + - + siUR - - + + - - siRac 1 - - - - + + ib: Rac ib: p-Pak Thr 423 ib: p-Pak Ser 199 ib: p-LIMK ib: p-Akt Ser 473 ib: Actin Lellean JeBailey

  46. Insulin signalling pathway Rac Actin dynamics Insulin Receptor P P IRS-1 P P P P p85 p110 PI 3,4,5-P3 PDK Akt AS160 GLUT4 translocation

  47. Insulin signalling pathway Insulin resistance inducers P P IRS-1 P P P P Insulin resistance inducers Rac Actin dynamics Akt Insulin Receptor p85 p110 PI 3,4,5-P3 PDK AS160 GLUT4 translocation

  48. Insulin signalling pathway P P IRS-1 P P P P Rac Actin dynamics Akt fusion arrival Insulin Receptor p85 p110 PI 3,4,5-P3 PDK AS160 GLUT4 translocation

  49. Thank you!

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