Pid1 Adipose Tissue Specificity, Such As The Construction Of Transgenic Mice And Phenotypic Analysis

Obesity is speeding to become an important public health issue globally. Alongwith the rapid development of economy and improvement of people’s life standard, itis also getting more and more pervasive in China. It has already drawn attentions ofpediatricians all over the world that the incidence of obesity among children isclimbing up and that more type2diabetes cases have been diagnosed on youngerages. Researches on obese children and adolescents in China also indicate raises ofinsulin-resistant factor and abnormalities in glucose tolerance test (GTT), althoughthe cases diagnosed as type2diabetes are relatively limited. It should be pointed outthat reduced insulin sensitivity or insulin resistance are important events for type2diabetes at early stage, therefore the study of obesity and obesity in the pathogenesisof insulin resistance has become an important field of pediatric researches.In our earlier studies, we isolated and characterized PID1, a novel hunman genethat was expressed at a high level in the omental adipose tissue of obese patients. Thisgene was primarily high expressed in insulin-sensitive tissues, including skeletalmuscle, adipose tissue and heart. We also found that the expression of PID1mRNAcould be regulated by a variety of factors that are related to insulin sensitivity (such asTNFα、IL-6, et al). Our findings suggested that PID1might be a new candidate generelated to obesity-associated insulin resistance. Further studies have shown that PID1can regulate insulin sensitivity of fat cells through the IRS1-PI3K-AKT insulin signaltransduction pathway. However, all studies so far are limited to in vitro level,therefore we generated a PID1fat-specific expression transgenic mouse model tofurther study the effect of PID1on the development of obesity, glucose and lipidmetabolism, and insulin sensitivity as well as the underlying mechanism. Part ⅠGeneration of adipose tissue-Specific PID1Expression Transgenic MiceObjective: Establishment of adipose tissue-specific PID1expression vectorpL253-aP2-PID1, generation of adipose tissue-specific PID1expression transgenicmice.Methods: PID1gene was cloned from mouse with RT-PCR and identified withsequencing analysis. PID1gene was inserted into the down stream of ap2promoterwhich expresses at adipose tissue specially to construct the vector pL253-aP2-PID1.The pL253-aP2-PID1construct was linearized using Pvu1and injected into pronucleiof fertilized eggs from the mice. The genotype of transgenic line was identified byPCR and the expression level of the gene PID1was determined by Western Blot.Results:1) Adipose tissue-specific PID1expression vector pL253-aP2-PID1wassuccessfully constructed;2) Two independent lines with PID1overexpression inadipose tissue were obtained.Conclusion: The adipose tissue-specific PID1expression transgenic mice wereobtained.Part ⅡAnalysis the phenotype of the adipose tissue-specific PID1expressiontransgenic mice on a standard chow dietObjective: To observe the development of obesity, glucose and lipid metabolism,and insulin sensitivity of the adipose tissue-specific PID1expression of transgenicmice on a standard chow diet.Methods: The adipose tissue-specific PID1expression transgenic mice and thesame age and sex wild-type mice were weight from21days after birth each two weeks; H&E staining was used to observe the morphology of the adipose tissue andliver tissue; a metabolic cage experiment was used to observe the feeding,thermogenesis, oxygen consumption, CO2generation, respiratory quotient andactivity; the changes of the plasma biochemical parameters(glucose, cholesterol,triglycerides, HDL, LDL) were used to observe the glucose and lipid metabolism;serum insulin level, glucose tolerance tests and insulin tolerance tests were used toview the insulin sensitivity.Results:1) Body weight was no significantly difference; H&E staining found thesize of fat cells was no significant change;2) the size of lipid droplets in liver are nosignificant difference;3) feeding, oxygen consumption, thermogenesis, CO2producedand respiratory quotient was no significant difference, but the activity of thetransgenic mice was significantly increased;4) the plasma glucose, cholesterol,triglycerides, LDL and HDL levels were no significant difference;5) the seruminsulin level, glucose tolerance and insulin tolerance were no significant difference.Conclusion: The adipose tissue-specific PID1expression transgenic mice were fedon a standard chow diet had no significantly changes about the obesity phenotype, theglucose and lipid metabolism and insulin sensitivity.Part ⅢAnalysis the phenotype of the adipose tissue-specific PID1expressiontransgenic mice on a high fat dietObjective: To observe the development of obesity, glucose and lipid metabolism,and insulin sensitivity of the adipose tissue-specific PID1expression of transgenicmice on a high fat diet.Methods: The adipose tissue-specific PID1expression transgenic mice and thesame age and sex wild-type mice were weight and fed on a high-fat diet from2 months after birth weekly; H&E staining was used to observe the morphology of theadipose tissue and liver tissue; the changes of the plasma biochemical parameters(glucose, cholesterol, triglycerides, LDL, the plasma triglyceride clearance) wereused to observe the glucose and lipid metabolism; serum insulin level, glucosetolerance tests and insulin tolerance tests were used to view the insulin sensitivity;Immunoblotting and immunofluorescence was used to measure the phosphorylationand total protein contents of the IRS1-PI3K-AKT insulin signal transduction pathwayand LRP1.Results:1) Body weight had a significantly increased after10weeks of a high-fatdiet; the size of the fat cells are also significantly increased;2) the lipid droplets inliver had a significant rise;3) the plasma triglycerides had a significant increase, butthe plasma glucose, cholesterol and LDL levels were no significant difference;4) theplasma triglyceride clearance showed a significant decrease;5) the plasma insulinlevel was no significant difference, glucose tolerance was significantly differences at90minutes and insulin tolerance had a significantly weakening at15minutes;6)Western Blot and immunofluorescence analysis of fat tissue have shown thatinhibition of IRS1-PI3K-AKT signaling pathway, and LRP1expression wassignificantly increased.;Conclusion: The adipose tissue-specific PID1expression transgenic mice occur anobesity phenotype, have disorders of carbohydrate metabolism and lipid metabolism;the decreased insulin sensitivity on a high fat diet; this change may be due to PID1overexpression inhibited IRS1-PI3K-AKT signaling pathway.