Molecular Mechanisms Regulating Hepatic Fetuin-A Expression
Type of DegreePhD Dissertation
DepartmentNutrition, Dietetics and Hospitality Management
MetadataShow full item record
Fetuin-A (Fet-A) is a hepatokine, known to inhibit insulin signaling and is associated with diabetes, cardiovascular outcomes and inflammation. Post-translational modification of fetuin-A at different glycosylation and phosphorylation sites considered to regulate protein expression levels, stability, and biological activity. Previously, it has been suggested that phosphorylation status of Fet-A is critical to inhibit insulin receptor tyrosine kinase activity and downstream insulin signaling. However, certain factors related to its regulation are still unclear. Here we demonstrated that, recombinant Fet-A containing phosphorylation at the site of Ser312 impaired insulin signaling (AKT, MAPK, GSK-3β, and insulin receptor phosphorylation) and action on gluconeogenic enzyme (PEPCK gene expression) and glucose production in HepG2 cells. We have also shown that glucose and free fatty acid (palmitic acid) upregulate expression or secretion of Fet-A and its phosphorylated (pFet-A) forms in human hepatoma HepG2 cells. This upregulation of high glucose-induced Fet-A and pFet-A expression was associated with impairment of insulin signaling. On the other hand, insulin and AMP-activated protein kinase (AMPK) activation downregulate the high glucose induced Fet-A and pFet-A expression. To further understand the negative regulation by AMPK activation, we examined the effect of AMPK activator, AICAR, on Fet-A expression in HepG2 cells, Hep3B cells and primary rat hepatocytes. We observed that treatment of AICAR, signiﬁcantly down-regulated high glucose-induced Fet-A expression without affecting pFet-A expression. Effect of AICAR was associated with activation of AMPK, while inhibition of AMPK activation prevented AICAR-induced downregulation of Fet-A expression. In further exploration of downstream target of AMPK, we observed that AMPK-p38 MAPK axis play a critical role in the regulation of the hepatic Fet-A expression. Further, we demonstrated that short term effect of AICAR on Fet-A expression was mediated by proteosomal degradation. While long term treatment of AICAR is associated with decrease in hepatic expression of C/EBP beta, an important transcription factor involved in Fet-A regulation. Fet-A is classified as an acute phase protein, divergently regulated during injury and infection. Early inflammatory mediators negatively regulate, while late inflammatory mediators positively regulate Fet-A expression. To understand the differential effect on Fet-A during inflammation, we explore the effect of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) on Fet-A and pFet-A expression. Here we propose that Fet-A synthesis and secretion is upregulated with LPS treatment acutely (4hr). While, long term treatment with LPS downregulate Fet-A synthesis as described previously probably due to feedback regulation (24hr). On the other hand, treatment of TNF-α decreases hepatic Fet-A and pFet-A synthesis/secretion. This contradicting effect on Fet-A and pFet-A after 4hr treatment of LPS and TNF-α was observed due to increase and decrease of C/EBP-β expression respectively. Taken together, our study paves the way in understanding of the critical players involved in the regulation Fet-A, a hepatokine associated with insulin resistance, diabetes, and cardiovascular outcomes.