Construction Of Calcium Binding Protein S100A16 Knockout Mice And Effect Of S100A16 On Glucose And Lipid Metabolism In Obese Mice Induced By High Fat Diet And Its Mechanism

Objective:To establish the Calcium-binding protein S100A16 gene knockout mice model,which can be used for the study of S100A16,s biologic function.Diet induced obesity model(DIO)was setted up by high fat diet.To construct an insulin resistance model by HepG2 cell,a tool for further study of the biological function of S100A16.Methods:Step 1:To establish the S100A16 gene knockout mice model via Cre/loxP system.polymerase chain reaction(PCR)was used to identify the genotype of the offspring,the transcription level of S100A16 mRNA was detected by real-time quantitative PCR(RT-PCR),the expression of S100A16 protein was verified by Western blot(WB).Step 2:After S100A16 knockout mice(heterozygous mouse,S100A16KO+/-)been successfully constructed,S100A16 KO+/-mice and normal C57BL/6(B6,wild type,WT)mice were selected for the study.The subjects were subjected to high fat diet to construct a diet-induced obesity model,no high fat diet as the control group.Five weeks-old male mice were randomly divided into normal diet group(No high fat)and high fat diet group.There are four groups:① normal control group(NFD WT),n = 10,②B6 high fat group(HFD WT),n = 10,③S100A16KO+/-normal fat diet group(NFD +/-),n=10,④S100A16KO+/-high fat group(HFD +/-),n = 10.Mice were fed for 13 weeks and recorded weekly for weight,randomized blood glucose.Intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(ITT)was performed at appropriate time.At the time of 18 weeks,we completed this experiment,and tissues were taken from the mice body.The serum levels of triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL),low density lipoprotein(LDL)and other lipid metabolism related biochemical indices were measured.The total weights of visceral fat,such as epididymis and kidney,were recorded.Pathological changes were observed by pathologic staining.The transcriptions and translations of S100A16 and related gene in fat and liver tissues were detected by RT-PCR and WB,to explore how S100A16 works and its related mechanism.At the same time,the cell model was established.Insulin resistance was induced by 10-7μmol/L insulin in human HepG2 hepatocellular carcinoma cell,to research the relationship between insulin resistance and S100A16.Results:(1)S100A16 gene knockout heterozygote(S1O0A16KO+/-)mice could successfully grow and breed.There was no difference in the birth rate of male and female.While,so far,gene knockout homozygous mice(KO-/-)is not being found.The levels of S100A16 transcription and protein expression were significantly decreased in fat,skeletal muscle,liver,myocardium and lung tissue of S100A16KO+/-mice.(2)In the DIO model,there was no significant difference in body weight,fat weight,TQ TC and LDL between the NFD S100A16KO+/-group and the normal control group.Compared with the NFD group,the body weight,fat weight,TG,TC and LDL were significantly increased in the high fat diet group,and S100A16,Wnt and β-catenin were upregulated.Compared with WT high fat group,body weight,fat weight and TG were significantly decreased,S100A16,Wnt and P-catenin were down-regulated and HDL was up-regulated.(3)The model of insulin resistance was successfully established with human HepG2 cell,the expression of IRS-2 was down-regulated and S100A16 was up-regulated.Conclusion:(1)The S100A16 knockout mice model was successfully established,an animal model to study the biological function and molecular mechanism of S100A16 in further.(2)S100A16 is closely related to the increase in body weight and the accumulation of fat,and S100A16 may regulate obesity by affecting the Wnt/β-catenin pathway.(3)S100A16 is associated with insulin resistance.S100A16 is widely expressed in the body,having a variety of biological functions.homozygous mice having not been found may suggest that S100A16 is associated with reproduction or embryonic teratogenesis.The next step,we can block the pathway at the cellular level to observe the changes of S100A16,using ICG-001,a specific blocker of the Wnt/β-catenin pathway.At the same time,high-throughput techniques(gene chip,mass spectrometry,etc.)can be used to screen the substrate.S100A16 is associated with obesity and insulin resistance,but its specific molecular mechanisms need to be explored in the further.