The Roles Of The Tissue-specific Overexpression Of MiR-155 In Mice In Regulating Blood Glucose

Background and objectiveDiabetes is a complex,chronic disease that is influenced by environmental and genetic factors and leads to severe complications such as end-stage renal failure,cardiovascular diseases,blindness and amputation.However,the major mechanisms underlying the pathogenesis of diabetes remain unclear.Nowadays,diabetes is a worldwide epidemic and the rate of increase in its prevalence in developing countries is alarming,which emphasizes the urgent need to explore new pathogenesis of diabetes and effective treatment strategies.Micro RNAs(miRNAs)are a class of small non-coding RNA molecules involved in various physiological and pathological processes.As a multifunctional miRNA,miR-155 plays crucial roles in regulating glucose and lipid metabolism as well as insulin sensitivity.Furthermore,in a pilot experiment,we found that miR-155 levels in serum of type 2 diabetes(T2D)patients were lower than in healthy subjects.However,its molecular mechanisms in glucose metabolism,insulin sensitivity and the pathogenesis of diabetes are poorly understood.Furthermore,emerging evidence shows that miRNAs have been implicated in glucose metabolism and insulin sensitivity by targeting insulin-sensitive tissues such as liver,skeletal muscle and adipose tissue.In order to investigate the function of miR-155,this study aimed to explore the roles of miR-155 in regulating glucose metabolism and insulin sensitivity by using liver-specific overexpression transgenic mice(Rm155LG/Alb-Cre mice)and muscle-specific overexpression transgenic mice(Rm155LG/Ckmm-Cre mice).Methods1.Screening Rm155 LG transgenic miceRm155LG mice were determined by m RFP assay by using the Xenogen IVIS Lumina Imaging System and stereo fluorescent microscope at birth,confirmed by PCR-based genotyping.2.Generation of Rm155 LG transgenic mice with muscle-specific overexpression Rm155LG/Ckmm-Cre double transgenic mice(Rm155LG/Ckmm-Cre mice)were generated by mating Rm155 LG mice with Ckmm-Cre mice.Rm155LG/ Ckmm-Cre mice were determined by Luc assay,and the expression of miR-155 transgene in the muscle of Rm155LG/ Ckmm-Cre mice was detected by qRT-PCR.3.Investigating the roles of miR-155 in glucose metabolism1)Comparison of blood glucose concentrations in fed-state,12-hour–fasted and 24-hour–fasted mice.2)qRT-PCR and Western blot were used to detect the expression of miR-155 and genes involved in glucose metabolism in wild-type C57 BL /6 mice.4.Investigating the effects of miR-155 on glucose metabolism and related mechanisms by using Rm155LG/Alb-Cre or Rm155LG/Ckmm-Cre mice1)Intraperitoneal glucose tolerance test(IPGTT),insulin tolerance test(ITT)and pyruvate tolerance tests(PTT)were performed on control and Rm155LG/Alb-Cre or Rm155LG/Ckmm-Cre mice2)qRT-PCR and Western bolt were used to detect the expression of genes involved in glucose metabolism in Rm155 LG/Alb-Cre mice.Results1.miR-155 and Luc transgenes were successfully activated in a muscle-restricted pattern in Rm155LG/Ckmm-Cre mice,as confirmed by Luc assay.2.Fasting state blood glucose levels were lower than fed-state and refed-state in C57 B L/6 male mice.qRT-PCR and Western blot analysis showed that Pck1,G6 PC and PPARGC1 A expressions were increased in fasting state in the liver.Also,qRT-PCR analysis showed that the expression of miR-155 decreased in fasting state.3.Fasting and fed-state blood glucose levels were lower in Rm155LG/Alb-Cre male mice.Glucose tolerance tests(GTTs)indicated that Rm155LG/Alb-Cre mice cleared glucose more efficiently than controls.Insulin tolerance tests(ITTs)indicated that Rm155LG/Alb-Cre mice were more sensitive to insulin than controls.Pyruvate tolerance tests(PTTs)indicated that hepatic glucose output was reduced in Rm155LG/Alb-Cre mice.4.GTTs indicated that Rm155LG/Ckmm-Cre mice cleared glucose more efficiently than controls.ITTs indicated that Rm155LG/Ckmm-Cre mice were more sensitive to insulin than controls.5.Rm155LG/Alb-Cre mice demonstrated decreased hepatic expression of genes involved in gluconeogenesis,glycolysis,TCA cycle and pyruvate metabolism by qRTPCR analysis.Western blot analysis showed that Pck1,G6 PC and PPARGC1 A expression were decreased in Rm155LG/Alb-Cre.Conclusions1.miR-155 transgene was successfully overexpressed in a muscle-restricted pattern in Rm155LG/Ckmm-Cre mice by Cre/lox P system.2.Either liver-specific or muscle-specific overexpression of miR-155 in mice leads to improved glucose tolerance,insulin sensitivity and impaired gluconeogenesis,which suggested that miR-155 may contribute to the glucose homeostasis and could be a potential target in diabetes.