טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRozentzweig Dafna
SubjectRole of AHNAK in the Mechanism of Insulin Resistance:
Implication on GLUT4 Gene
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Eddy Karnieli
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

 Background and Aims: Obesity and type 2 diabetes (DM2) are associated with reduced GLUT4 gene expression and function, hyperlipidemia and insulin resistance. Free fatty acids (FFA) modulate gene transcription by exerting a direct, membrane-independent influence gene expression, mRNA stability and cellular differentiation.

We have previously shown that high levels of arachidonic acid (AA) repress GLUT4 promoter activity via specific promoter region (AA-R) but the mediator remained elusive. Our aim was to identify the protein/s mediating GLUT4 gene repression upon chronic exposure to AA and examine their role in diabetes.

Methods and Results: Nuclear extracts from AA-treated H9C2 cardiomyocytes were subjected to GLUT4-P-biotin-avidin affinity column, using AA-R as a bait, followed by mass spectrometry analysis. This analysis detected AHNAK, a giant phosphoprotein   attached to GLUT4-P.  Data regarding AHNAK association with membranal calcium channels show that AHNAK could be involved in altering signaling pathways mediated via Ca and the extracellular matrix (ECM) in cellulo. Its nuclear/cytoplasmic localization is determined by AKT phosphorylation on Ser-5335.   

Transient co-expression in primary rat adipocytes (PRA) of expression vectors for either C-terminal, middle or N terminal parts of AHNAK, resulted in 60±5%, 70±3% and 80±3% (p≤0.05) repression of GLUT4-P activity, respectively; This was partially alleviated by insulin (100 nM, 24 hrs). Using 5’ deletion analysis of GLUT4-P we found that deletion of -2155/-1887 bp region led to partial alleviation of GLUT4-P repression mediated by AHNAK parts. An additional promoter region -675/-66 bp, contributed only to AHNAK middle part ability to repress the GLUT4-P.

Chromatin immunoprecipitation showed binding of AHNAK middle part to GLUT4-P. Silencing AHNAK expression in adipocytes, resulted in significant 2-fold increase in cellular Glut4 protein, and a protection from AA-induced depletion.  Hyperlipidemia (200uM AA, 24-48hrs) decreased AHNAK gene expression to 40% in PRA,while hyperglycemia (25mM, 24 hrs) increased it to 140%.

In cardiac and skeletal muscle obtained from STZ diabetic rats, AHNAK mRNA levels were increased 270- 290±30%  (p≤0.05), respectively, compared to control. Interestingly, upon 8 days insulin therapy (8IT) AHNAK mRNAs returned to control level. In contrast, AHNAK levels in rat primary adipocytes (PRA) were 2.5-fold decreased in STZ and 8IT did not alter its expression.

Conclusions: AHNAK represses GLUT4 gene expression, and contributes to lipotoxicity-induced insulin resistance. Our data introduce AHNAK as a novel regulator of GLUT4 gene expression and a potential molecular target for diabetes and obesity treatment.