Study On The Ubiquitination Degration Of KLF11 And Its Regulation Of Pancreatic β Cell Function

Since the beginning of the 21st century,with the continuous development of social economy and the improvement of people’s living standards,the prevalence of metabolic diseases such as diabetes,obesity,hyperlipidemia and fatty liver is rising rapidly.The further progression of these metabolic disorders can induce serious cardiovascular and cerebrovascular events,including atherosclerosis and stroke,seriously threatening people’s physical and mental health,and bringing heavy medical burden to social development.Diabetes is a group of chronic metabolic disorder syndrome characterized by hyperglycemia.In recent years,the morbidity and control of diabetes at home and abroad are in very severe situation.By 2013,the prevalence rate of diabetes in China have exceeded 12%.With the deepening research,the gene analysis technology for the etiology of diabetes is becoming more and more mature.It has been found that a variety of single gene mutations lead to abnormal structure or dysfunction of theβcell,thus inducing single gene diabetes.Maturity-onset diabetes in the young（MODY）is the most common type of single-gene diabetes.MODY usually develops from 6 months to 25 years old,far earlier than ordinary type 2 diabetes.It is characterized by mutations in autosomal genetic information and non-ketotic hyperglycemia caused by abnormal structure or dysfunction of pancreatic isletβcells.And it is usually not accompanied by characteristic symptoms of type 2 diabetes such as insulin resistance and obesity.Of the 14 subtypes of MODY that have been identified,MODYⅦdiabetes is closely related to the transcription factor KLF11.Current studies on the function of KLF11 have found that KLF11 plays a clear role in cancer suppression in lung cancer,pancreatic cancer,ovarian cancer,hematologic tumors and other malignant diseases.In the cancer cell mentioned above,the expression of KLF11is obviously abnormal.However,until now,the effect of KLF11 on the body’s blood glucose homeostasis has not yet been determined,and the regulation mechanism of its stability has not been reported.The elucidation of these issues can deepen the understanding of the pathogenesis of diabetes and identify the underlying factors affecting KLF11 expression.Through cell experiments in vitro,our group confirmed that overexpression of KLF11inhibited the transcriptional activity of insulin gene inβ-cell line like INS1 and MIN6 cells.Mutations in the binding sequence of KLF11 to the histone deacetylase complex Sin3A or inhibiting the activity of histone deacetylase,HDACs,in cells can effectively block the inhibitory effect of KLF11 on insulin gene.Moreover,interfering with KLF11 expression in INS1 cells can significantly enhance the expression of insulin genes Ins1 and Ins2.These results suggest that KLF11 has a significant inhibitory effect on insulin gene transcription in cell lines in vitro,via a HDACs recruiting way.Considering that KLF11 is expressed in various tissues of the whole body,in order to accurately investigate the function of KLF11 in pancreaticβ-cells,the Ins2-Cre model tool mouse constructed by ourselves in our laboratory was occupied to construct tissue-specific KLF11 knockout mice,namely,β-KLF11KO mice.Our data showed that the glucose tolerance of mice withβ-KLF11KO was significantly improved.In mouse primary islet cells,the m RNA expression level of insulin gene was significantly stimulated,indicating that KLF11 had an inhibitory effect on insulin synthesis in mice.However,there was no significant change in insulin levels in plasma or total insulin content,suggesting that there may be a compensatory mechanism that partially compensates for the biological effects of KLF11 knockout.Preliminary results suggested that KLF11 is a negative regulator of insulin synthesis.This paper analyzes the regulation mechanism of KLF11 function in detail.First,we reviewed the literature and found that E3 ubiquitin ligase FBW7 can ubiquitinate and degrade KLF5,a member of the same family.In vitro cytological tests confirmed that overexpression of FBW7 resulted in a significant decrease in KLF11 protein levels in INS1cells.The co-immunoprecipitation assay proved that FBW7 could recognize KLF11 and enhance its ubiquitination level.What’s more,after interfering with the expression of FBW7,the degradation rate of KLF11 slowed down significantly.These results all confirm that KLF11 is one of the substrate targets of FBW7,and its ubiquitination degradation process is regulated by FBW7.Subsequently,to investigate the effect of FBW7 on insulin synthesis in isletβcells,we constructed a tissue-specific FBW7 knockout mouse model to clarify the specific function of FBW7 in isletβcells.After successfully obtaining model mice,we analyzed the metabolic phenotypes ofβ-FBW7KO mice on a regular diet.Compared with wild-type littermate,β-FBW7KO mice ate and drank more food and water but lost weight.It was found that the fasting blood glucose and random blood glucose ofβ-FBW7KO mice were significantly increased,while the plasma insulin content was significantly decreased.The glucose tolerance was significantly impaired.And the insulin secretion function was significantly impaired.In a word,β-FBW7KO mice suffered serious symptoms of diabetes.Further analysis showed that there was no significant change in the islet structure and number of mice withβ-FBW7KO mice,and the proliferation and apoptosis levels of mouse cells were also normal.However,our results showed that the m RNA levels of Ins1and Ins2 were significantly decreased in mouse islet cells.And the total insulin content was significantly decreased.In order to rule out secondary changes in islet caused by hyperglycemia and developmental abnormalities that might result from FBW7 deficiency,we constructed a mouse model of tamoxifen-induced FBW7 knockout in mice with the Ins1-Cre^ER model.At the early stage of tamoxifen induction,the model mice showed no significant abnormal glucose metabolism.It took 9 days to start getting symptoms after being infected,including impaired glucose tolerance,dysfunction of insulin synthesis and controlled secretion,significantly reduced storage capacity.These results suggest that the absence of FBW7 in islet cells can lead to impaired insulin synthesis in a short period of time.At the same time,our proteins analysis of primary islet tissue in mice withβ-FBW7KO showed that the expression level of KLF11 increased significantly after FBW7 functionally deficient.These findings suggest that KLF11 accumulates in large quantities after FBW7 deficiency,the insulin synthesis and secretion of the cells involved are inhibited,and the insulin storage is significantly reduced.In conclusion,we used cytological experiments and a variety of mouse models to draw the following conclusions:（1）in islet cells,KLF11 has a negative regulatory effect on the transcription of insulin gene.（2）the ubiquitination and degradation of KLF11 is regulated by E3 ubiquitin ligase FBW7.（3）in isletβcells mice with FBW7 dysfunction,the stability of KLF11 is increased,leading to function enhancement,resulting in misfunction of insulin synthesis and absolute insulin deficiency inβ-FBW7KO mice,which suffered severe diabetic phenotype.