טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDabour Ziad
SubjectThiamine Effect on Insulin Responsive Glucose Transporter
(Glut4) Gene Expression
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Eddy Karnieli


Abstract

Glucose uptake into eukaryotic cells, under basal and insulin-stimulated conditions, depends on the function of glucose transporters GLUTs. The importance of tissue-specific regulation of GLUT4 expression for maintaining glucose homeostasis is amplified in altered metabolic states. Insulin resistance in Type 2 diabetes, obesity and aging has been associated with marked reduction in the cellular level of GLUT4 proteins, which leads to impaired insulin stimulation of glucose transport.

Thiamine is an important cofactor regulating enzymatic activity and cellular energy. Thiamine pyrophosphate is a cofactor for pyruvate, a-ketoglutarte and branched chain dehydrogenase complexes, and transketolase, thus regulating of glucose metabolism and pancreatic b-cell function. In yeast, it regulates expression of genes involved in thiamine metabolism, and regulates gene expression of transketolase and pyruvate dehydrogenase in eukaryotic cells. Recently, it was discovered thiamine-responsive megaloblastic anemia (TRMA) is associated a mutation in the thiamine transporter itself.  However, the contribution of thiamine to GLUT4 gene expression is not yet fully understood.  Therefore, we investigated the hypothesis that thiamine regulates GLUT4 gene expression and function in insulin target cells.

We found that after 3 weeks of thiamine deficiency in rats, levels of GLUT4 mRNA were 40-50% decreased in adipose tissue, while 2-fold increased in skeletal muscle, compared to control. One week of thiamine refeeding restored GLUT4 mRNA levels in adipocytes to 250% of control level, while no change was detected in muscle. At promoter level, we found that  thiamine (25 mM, 24-48 hrs) enhanced transcriptional activity from a GLUT4-LUC promoter reporter by 200% and 230%, in primary rat adipocytes and in CHO-K1 cells, respectively, while this activity was 30% repressed by pyrithyamine (thiamine inhibitor). Using 5`-deletion analysis, GLUT4 promoter region -1779/-1742 bp was revealed, that mediated its activation by thiamine and whose deletion rendered the promoter irresponsive to thiamine.  Electromobility shift assays further indicated that thiamine enhances the binding to this region, of as yet unidentified mediating protein(s), that is neither NFkB-p50, NFkB-p65, nor C/EBP-alpha.

In this work we have identified the GLUT4 gene as a molecular downstream target for thiamine. We have shown that thiamine activates transcription from GLUT4 promoter by enhancing the binding of as yet unidentified protein(s), to a specific promoter region -1779/-1742 bp. Thus we have identified a novel thiamine response element on GLUT4 gene.