Ph.D Thesis

Ph.D StudentBar-Yoseph Fabiana
SubjectMechanism of Action of NF-kB and IKK on the Expression and
Activity of Glucose Transporters in
Pathophysiological Metabolic States
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Eddy Karnieli


Background and aims: Inflammatory signaling pathway has been suggested to induce insulin resistance in Type 2 Diabetes Mellitus (DM2) and obesity by inhibiting Insulin-IRS1-PI3 kinase pathway. Inhibiting IkappaB kinase (IKK) activity was shown to increase insulin sensitivity. Therefore, we examined the hypothesis that the insulin-responsive glucose transporter (GLUT4) is a direct molecular target for IKK- NF-kB transcriptional complex leading to the decrease in cellular glucose uptake.

Methods and results: C2C12 myotubes and HEK-293 cell-lines were transiently co-transfected with luciferase-conjugated GLUT4 promoter reporter and expression vectors for either IKK or NF-kB subunits. Over-expression of IKK (IKKalpha and IKKbeta) dose-dependently repressed GLUT4 promoter transcriptional activity to 60% of basal level. This effect was abolished in presence of salicylates (10mM, 48hr). Co-expression of NF-kB p50/p65 heterodimer dose-dependently decreased GLUT4 promoter transcriptional activity by 70% and 25% in HEK and C2C12 myotubes. Co-expression of p50 subunit increased the GLUT4 promoter transcriptional activity by ~200%, while p65 alone repressed it by ~50%. 5’-deletion analysis revealed specific DNA regions on the GLUT4 promoter that might mediate the different NF-kB effects. Measuring p50 and p65 protein levels in muscle biopsies obtained from diabetic and normal control patients showed a marked reduction of p50 subunits to ~50% of control level but no significant change in p65 levels. Using specific siRNA, p65 protein subunit was reduced to ~30% compared to control level followed by 150% elevation in cellular GLUT4 protein level.

Conclusion: Our data suggest that NF-kB subunits are regulators of the GLUT4 gene transcription. Since p65 suppression resulted in significant increase of cellular GLUT4 protein level, modulation of IKK- NF-kB pathway is a potential target for type 2 diabetes therapy.