טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShitrit Nurit
SubjectThe Role of FOXO1 Transcription Factor in Regulating
Glucose Transporter 4(GLUT4) Gene Expression
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Eddy Karnieli


Abstract

Background: Forkhead box O (FOXO) transcription factors are downstream targets for insulin. The role of human FOXO1 in regulating gene expression of insulin-responsive glucose transporter (GLUT4) is unknown. We examined whether FOXO1 regulates GLUT4 gene expression, using FOXO1 over expression on one hand, and FOXO1 gene silencing on the other.


Results: Using siRNA technique we show that specific inhibition of FOXO1 mRNA by 80%, induced significant 120% increase in GLUT4 protein level. Transient co-expressions of FOXO1 in GLUT4-expressing HEK cells, repressed transcriptional activity of GLUT4 promoter reporter by 50%. FOXO1 trans-activated transcription of a synthetic IRE-Luc promoter reporter by up to 30 fold. Under these conditions, immunofluorescent staining showed that FOXO1 protein is mainly localized to the nucleus.

Co-expression of the GLUT4 promoter reporter with FOXO1 mutants H215R (DNA Binding Domain defective), or AAA (triple phosphorylation defective), significantly enhanced the transcription activity of GLUT4 promoter up to 200% and 300% of basal level, respectively. Under these conditions both mutants localized to the nucleus. Insulin incubation for 24 hours had no effect on the promoter activity of GLUT4 in the presence of wild type FOXO1or H215R mutant. Preincubation with insulin for 24 hours induced a redistribution of the wild type FOXO1 and H215R mutant to the cytoplasm, while affecting neither the AAA trans-activation effect nor its nuclear localization. 5’-deletion analysis of the GLUT4 promoter revealed a

-675/-66 bp region that might mediate human FOXO1 repression of the transcriptional activity  of GLUT4 promoter.


Conclusion: Taken together, we show that GLUT4 gene expression is down-regulated by native human FOXO1. These effects require both intact DBD and phosphorylation sites. Since inhibiting FOXO1 expression or activity leads to increase in GLUT4 cellular content, FOXO1 is suggested as a potential target for therapy in Type 2 Diabetes Mellitus.