1. Study on mTOR-regulated GLUT4 Translocation
Mabina M. Kiawu, Pi-I Debby Hsu, and Dr. Do-Hyung Kim
Department of Biochemistry, Molecular Biology and BiophysicsUniversity of Minnesota, Twin Cities, MN 55455
Materials and Methods
Results
Abstract
A PRP1 gene was clone into a pLKO silencing plasmid vector. Then, GLUT4 was labeled with GFP gene. Sh-PRP1, GFP-GLUT4 along with FuGene6 reagent were transfected into HepG2 cells, which had grown in DMEM at 37ºC. The transfected cells were starved for 24 hours, and serum was added to both the control and experimental groups. Five minutes prior to confocal microscope analysis, insulin was added to experimental groups.
mTOR (mammalian target of Rapamacin) is a Serine/Threonine kinase, and it is a downstream protein of Phosphatidylinositol- 3-kinase/AKT in the glucose metabolism pathway that is used by cells to sense nutrients in their environment. mTOR is activated by AKT (Protein Kinase B), which leads to gene expression and protein synthesis, and glucose metabolism. Abnormality in PI3K-AKT-mTOR signaling pathway contributes to pathogenesis of several human diseases such as diabetes and cancers. Experiments have shown that insulin stimulation of Iinsulin Receptor Tyrosine Kinase leads to the phosphorylation and activation of an intracellular protein, IRS-1. This protein phosphorylates and activates PI3K, which then activates other downstream proteins (AKT) leading to mTOR activation and GLUT4 translocation. However, activation of this pathway by PDGF or IL-4 does not necessarily lead to GLUT4 translocation even though PI3K is activated in the process. Therefore, it is suspected that another protein must be involved in the insulin-stimulated GLUT4 translocation. Experiments have shown that when mTOR is inhibited by Rapamycin, there is a 50% reduction in GLUT4 translocation. When both PI3K and mTOR are inhibited, the cell becomes insensitive to signal transmitted by IRS-1 and so no GLUT4 translocation occurs. To determine if mTOR is the downstream protein of PI3K-AKT involved in glucose metabolism, we cloned Sh-DNA’s into a pLKO vector, which expressed an sh-RNA (an RNAi) that silenced the expression a PRP gene, inhibiting the synthesis of mTOR component protein, PRP (Proline-rich-protein). As expected, when the PRP gene expression is silenced, the activity of mTOR is inhibited, and there is a significant reduction in GLUT4 translocation. These data suggest that mTOR is a very important downstream protein of PI3K-AKT signaling pathway involved in glucose metabolism.
Glucose
mTOR
Insulin
GLUT4 translocati on
Normal Conditio n +
Control group -
Experime ntal Group
“Labeled” GLUT4 gene with GFP +
Design gene to
inhibit mTOR activity
Transfect
cell
Introduction
mTOR
Observe Change Using Confocal Miscroscope
Mammalian Target For Rapamacin (mTOR) is a Serine/Threonine Kinase that cells use to sense nutrients in their surroundings. Activation of mTOR by different signal molecules can lead to gene transcription, protein synthesis and cell proliferation.
shRNA Construction
From literature review, under normal condition, when mTOR is not inhibited, GLUT4 translocation occurs and the number of GLUT4 in the plasma membrane increases 2-3 fold 10 minutes after insulin stimulation. In the control groups, in which insulin is absent and mTOR is inhibited, no GLUT4 translocation is observed: a characteristic basal state. In the experimental groups, in which insulin is present but mTOR is inhibited, cells still behave as basal state and no GLUT4 translocation is observed.
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)
GLUT4 Translocation
Conclusion
GLUT4 is an isoform of glucose transporters in muscles, fats and liver cells. Under basal condition, GLUT4 is fully sequestered in intracellular compartment. Upon insulin stimulation, target exocytosis of GLUT4 to the plasma membrane and restrained transport endocystosis participate to increase glucose uptake. This movement of GLUT4 is called GLUT4 translocation.
When mTOR is inhibited:
Some cells support our hypothesis by showing a reduction in GLUT4 translocation. However this was
not true for all transfected cells possibly due to diffrences in reaction condition, such as temperature, tranfection method, contamination, and etc. Therefore, more research need to be done to confirm our hypothesis for sure. According to preliminary results, mTOR is an important downstream protein of PI3K-Akt signaling pathway involved in glucose metabolism.
mTOR-GLUT4 Interaction
When insulin binds to Insulin Receptor Tyrosin Kinase, the receptor dimerizes and crosss phosphorylates its cytosolic domain. Upon cross phosphorylation, the cytosolic domain of the receptor is activated and serves as an activation site for many proteins including Insulin Receptor Substrate (IRS). IRS phosphorylates PI3K (phosphoinositide- 3-kinase), which also phosphorylate Akt (protein Kinase B), which then phosphorylates and activates mTOR and also regualates GLUT4 translocation. However, activation of this same pathway by PDGF does not lead to mTOR stimulation and therefore does not lead to GLUT4 translocation. Since mTOR is activated when insulin binds to this pathway, we hypothesize that mTOR is the protein involved in GLUT4 translocation.
Future Directions
Finding other possible proteins involved in stimulating GLUT4 translocation
Sequencing more mTOR component proteins and novel proteins that bind mTOR and affect its activaties
Studying GLUT4 interaction with the plasma membrane
Studying the involvement of both mTOR and GLUT4 translocation in type II diabetes
Confocal Microscope
Acknowledgments
high resolution
3D reconstruction
Blur-free image of thick specimen at different depth ?
We thank Dr. Do-Hyung Kim for providing the resources and insights to this project. We would also like to extend our appreciations to Emilie Vander Haar and Seong-Il Li for their supports and encouragements to this project.
References
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