|Ph.D Student||Karni Shiri|
|Subject||NFkB Regulates GLUT4 Expression and Function in Insulin|
|Department||Department of Medicine||Supervisor||PROFESSOR EMERITUS Eddy Karnieli|
|Full Thesis text - in Hebrew|
The NF-kB pathway is activated in response to inflammatory stimuli; it can induce insulin resistance. The present study examines the hypothesis that GLUT4 gene expression and function is a molecular target for the NF-kB signal transduction pathway.
In primary rat adipocytes (PRA), streptozotocin (STZ) induces diabetes was associated with increased expression of both NF-kB (p50,p65) sub-units by 2 fold compared to controls. Insulin therapy normalized NF-kB mRNA levels. In contrast, the GLUT4 mRNA level was decreased in STZ diabetic rats, while insulin therapy enhanced its expression to higher than normal levels.
In cardiac muscle biopsies obtained during elective coronary surgery, we found that type 2 diabetes mellitus (DM2) was associated with 20% increased and 20% decreased levels of p65 and GLUT4 mRNA, respectively.
Using GLUT4 promoter reporter co-expressed with NF-kB subunits in PRA and in L6 myotubes, we observed dose dependent repression, compared to basal levels. 5’-deletion analysis of GLUT4-P revealed two distinct promoter regions mediating the effect of p50 and p65. Chromatin Immuno-Precipitation (ChIP) indicated that p50 indeed binds to the GLUT4 promoter. Silencing of p50 and p65 in PRA enhanced the Glut4 protein level.
Glut4 function was assessed by translocation assay in L6-myc myoblasts. In basal state p65, transfection increased Glut4 translocation to the plasma membrane, similar to the insulin effect. Insulin stimulation further enhanced the translocation efficiency. Silencing NF-kB subunits significantly reduced Glut4 translocation in basal conditions. Further, silencing p50 subunits, with or without p65, impaired insulin stimulatory effects on Glut4 translocation to the cell surface.
In our search for the missing link between the NF-kB and the translocation pathways, we found that NF-kB overexpression resulted in augmented mRNA and protein levels of 14-3-3β, while silencing significantly decreased 14-3-3β mRNA levels. Co-IP experiments revealed augmented interaction between 14-3-3β and AS160 after overexpression of each NF-kB subunit separately and both of them together.
This study shows that GLUT4 is a molecular target for the NF-kB signalling pathway at its transcriptional and functional levels. On one hand, NF-kB subunits repress GLUT4 gene transcription in insulin target tissues. On the other hand, NF-kB upregulates Glut4 translocation machinery by enhancing 14-3-3 cellular level, and its interaction with AS160. thus improving insulin sensitivity. Overall, NF-kB modes of action presented in this study are potential targets for type 2 diabetes therapy.