Study on mTOR-regulated GLUT4 Translocation Mabina M. Kiawu and Pi-I Debby Hsu Dr. Do-Hyung Kim, Department of Biochemistry, Molecular Biology and Biophysics University of Minnesota, TC

What is mTOR? mammalian target for Rapamacin http://www.novartisoncology.com/images/rad001Large1.jpg

What is the specific metabolic pathway for mTOR? Rheb TSC1-TSC2 Insulin/ IGF-I ? Akt PI3K PTEN LY AMPK S6K1, 4E-BP1 protein synthesis, metabolic gene transcription, autophagy, IRS-2/IRS-1 Figure 1. A diagram showing interactions between components of the mTOR signaling pathway and negative regulation of insulin signaling by the mTOR pathway. Insulin Receptor Tyrosine Kinase IRS (insulin receptor substrate) PI3K (phosphatidylinositol-3-kinase) Akt (Protein Kinase B) mTOR(Serine/Threonine kinase)

Why is mTOR important? Abnormality in PI3K-Akt-mTOR signaling pathway contributes to pathogenesis of several human diseases Diabetes Cancers

What is GLUT4 Translocation?

Is GLUT4 translocation always regulated by PI3K-Akt pathway? InsulinIRSPI3KAktactivate mTOR and GLUT4 translocation BUT IL-4 or PDGFPI3KAktcell proliferation ≠> GLUT4 translocation http://www-ermm.cbcu.cam.ac.uk/fig003sfn.gif

Why is mTOR important in GLUT4 translocation? mTOR inhibited by Rapamycin50% reduction mTOR + PI3K inhibited cell insensitive to IRS signalno translocation

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How to test mTOR is the downstream protein of PI3K-Akt involved in glucose metabolism? + Design gene to inhibit mTOR activity “Labeled” GLUT4 gene Transfect cell Observe Change

What is shRNA? sense and antisense sequences from a target gene Short hairpin RNA sense and antisense sequences from a target gene makes a tight hairpin turn and can be used to silence gene expression http://www.panomics.com/SR1000.cfm

How to design a silencing plasmid? PRP1 (proline-rich protein 1) is a component protein of mTOR Cloning PRP1 gene into a shRNA vector (pLKO) Forward oligo 5’ CCGGT-(PRP1 sense)-CTCGAG-(antisense PRP1)-TTTTTG-3’ Reverse oligo 5’ AATTCAAAAA-(sense PRP1)-CTCGAG-(PRP1 antisense)-3’ (cloned in between AgeI and EcoRI sites in pLKO) http://www.addgene.org/pgvec1?f=c&identifier=8453&cmd=findpl

How can GLUT4 translocation be observed? GFP (Green Fluorescence Protein) label GLUT4 with GFP

How to transfer DNA into cell? Transfection FuGENE 6 (transfection reagent) GFP-GLUT4 shRNA vector HepG2 cell grown in DMEM

How to observe GLUT4 translocation? Confocal microscope high resolution 3D reconstruction Blur-free image of thick specimen at different depth http://www.leedsmicro.com/confocal/FV1000.jpg

How is GLUT4 translocated? From literature review (Thong, Dugani, and Klip, 2005) Unstimulated fat or muscle cells 3-10% (cell surface) >90% (intracellular compartments) Insulin stimulated Perinuclear GLUT4 conical cluster  tighter ring Surface GLUT4 increase 2-3 fold after 10 min Figure 1. Insulin regulation of glucose transporter GLUT4 distribution. from Thong, Dungani, and Klip, 2005, p272.

How is GLUT4 tranlocation affected when mTOR is inhibited? mTOR inhibited + no insulin stimulated expected basal state shPRP1 (basal state) mTOR inhibited +insulin stimulated still behave like basal state No shift of GLUT4 to the plasma membrane No increase of GLUT4 around nucleus No increase of surface GLUT4 shPRP1 (insulin stimulated)

Summary of Result Glucose mTOR Insulin GLUT4 translocation + -

Conclusion: When mTOR is inhibited: Some cells support our hypothesis  Reduction in GLUT4 But this is not true for all transfected cells. Why? Reaction condition (temperature, tranfection method, contamination, etc) More research need to be done to confirm our hypothesis for sure mTOR is an important downstream protein of PI3K-Akt signaling pathway involved in glucose metabolism, according to preliminary result.

Reference Farah S. L. Thong, Chandrasagar B. Dugani, and Amira Klip, “Turning On and Off: GLUT4 Traffic in the Insulin-Signaling Highway”, Physiology, vol. 20: page 271-284, 2005

Questions?