1. 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

2. What is mTOR? mammalian target for Rapamacin

3. 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)

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

5. What is GLUT4 Translocation?

6. 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

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

8. ?

9. 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

10. 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

11. 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)

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

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

14. How to observe GLUT4 translocation?
Confocal microscope
high resolution
3D reconstruction
Blur-free image of thick specimen at different depth

15. 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.

16. 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)

17. Summary of Result
Glucose
mTOR
Insulin
GLUT4 translocation + -

18. 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.

19. 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 , 2005

20. Questions?