The Role And Mechanism Of Ubiquitin Ligase FBXW7 In The Regulation Of Hepatokine-Fetuin A Protein Stability

With the rapid development of global economy and great changes of diet structure and lifestyle,the prevalence of obesity and type 2 diabetes has been growing year by year.In addition,obesity and type 2 diabetes are strongly associated with several cardiometabolic abnormalities,including,coronary heart disease,non-alcoholic fatty liver disease(NAFLD)and hypertension.Without doubt,obesity and its associated metabolic disorders have become an increasing severe public health problem worldwide.Therefore,it is an urgent for scientists and clinicians to be devoted to researches on obesity,insulin resistance and T2 DM.In recent years,growing evidences demonstrate that as a major metabolic organ,liver can synthesize and secret a variety of cytokines(called Hepatokines)to modulate glucose and lipid metabolism.And previous studiesshowed that ?2-HS-glycoprotein(also known as Fetuin A)is among the most important hepatokines in the regulation of glucose metabolism.Both animal and human studies have shown that Fetuin A is dramatically increased in obese individuals and consequently leads to insulin resistance and T2 DM.However,it remains largely unknown that the molecular mechanism of up-regulation of Fetuin A in obesity.In the present study,we would like to explore the mechanism from the aspect of Fetuin A's protein stability.Firstly,to determine the turnover course of Fetuin A protein,we transiently or stably transfected Fetuin A in HEK293 T or HepG2 cells respectively,which revealed that Fetuin A was rapidly degraded through ubiquitin-mediated proteasome system(UPS).Next,through amino acid sequence analysis,we found that Fetuin A contains the sequence(A – F – S – P – V – A – S),which could be recognized by FBXW7,an E3 ligase in UPS.Given the hypothesis that FBXW7 might be the E3 ligase of Fetuin A degradation,we overexpressed FBXW7 and Fetuin A in HEK293 T cells,and found that FBXW7 remarkably promotes Fetuin A degradation.Meanwhile,immunoprecipitation experiment also indicated that FBXW7 could target Fetuin A protein,increase its ubiquitination and subsequently accelerate its turnover.Furthermore,in order to elucidate the biological function of FBXW7-mediated Fetuin A degradation in obese mice,we determined expression levels of FBXW7 and Fetuin A in both ob/ob mice and HFD mice.A pronounced down-regulation of FBXW7 and up-regulation of hepatic,serum Fetuin A protein levels were observed.Therefore,we suppose that FBXW7 could play an important role in the regulation of Fetuin A in obesity.Then,ob/ob mice were infected with adenoviruses containing FBXW7 by means of tail vein injection,and liver tissues were collected after 10 days.As a result,hepatic overexpression of FBXW7 resulted in a significant decrease in both hepatic and serum Fetuin A levels,and a dramatic improvement in glucose metabolism.Similar results were also observed in high-fat-diet-induced obese mice.In conclusion,we demonstrated that FBXW7 negatively regulated Fetuin A protein level,and overexpression of FBXW7 in obese mice contributed to the improvement of glucose metabolism.Thus,our study revealed that FBXW7 play a role in the regulation of Fetuin A levels and insulin sensitivity.