| Glucose transporter 4(GLUT4)plays an important role in regulating glucose uptake in mammals.The research on GLUT4 is mainly focused on mammals,while the research on chicken GLUT4 is rare.In this study,according to the predicted sequence provided by NCBI,the full length of chicken GLUT4 gene was amplified,and the obtained sequence was analyzed by bioinformatics,and the spatial and temporal expression characteristics of GLUT4 were studied by RT-qPCR.Then,the response of chicken GLUT4 to exogenous stimulation was explored in vivo and in vitro.Finally,GLUT4 was overexpressed at the cellular level to explore the effects of GLUT4 gene on proliferation,differentiation,apoptosis and glucose uptake of chicken skeletal muscle cells,and RNA-seq was used to further explore the mechanism of GLUT4 gene function.Experiment 1: Cloning,sequence analysis and expression characteristics of chicken GLUT4 gene.In order to explore the sequence characteristics and expression characteristics of chicken GLUT4 gene,the chicken GLUT4 gene sequence was obtained by conventional PCR amplification,5’-RACE and 3’-RACE.And its genome structure,splicing pattern,subcellular localization prediction and homology were analyzed by bioinformatics.In addition,RT-qPCR was used to detect the distribution of GLUT4 in liver and striated muscle(myocardium,pectoralis and leg muscle)of chickens at 14 embryonic age(E14),E19,7 days of age(D7),D21 and D49.The results showed that the chicken GLUT4 gene produced at least 14 transcripts(Gen Bank accession number: OP491293-OP491306),encoding 12 amino acid sequences(length between 65-519 AA),and was predicted to have significantly different subcellular localization.These proteins contain a typical MFS domain.The main transcript(named as T1)consists of 11 exons,encoding 519 AA.In addition,the spatial and temporal analysis of chicken GLUT4 gene expression showed that chicken GLUT4 gene was mainly expressed in striated muscle,and the expression level of GLUT4 gene in chicken myocardium,pectoralis and leg muscle fluctuated significantly with the development of chicken.The expression level of GLUT4 gene in D21 was significantly higher than that in E14,E19 and D49(P <0.05).These data indicate that chicken GLUT4 undergoes complex alternative splicing events,and the expression level of GLUT4 in striated muscle is dynamically regulated as chickens develop.The results showed that these transcripts may play overlapping and different roles in chickens.Experiment 2 Effects of exogenous stimulation on GLUT4 gene expression in chicken.In order to explore the response of chicken GLUT4 to exogenous stimulation,this experiment was carried out in vitro and in vivo.In vivo experiments,5 IU / kg insulin solution,2 g/kg 10% glucose solution,2g / kg sodium pyruvate solution,30% energy restriction,15% energy restriction,15% protein restriction,fasting and other exogenous stimulation were used to detect the expression of GLUT4 mRNA in insulin-sensitive tissues.In vitro experiments,exogenous insulin,glucose,and starvation were used in myoblasts or SMSCs.The results showed that insulin,sodium pyruvate and feed restriction did not significantly change the expression of GLUT4 in vivo.At 60 min of glucose treatment,the expression of GLUT4 was significantly lower than that of the saline control group(P < 0.05).The expression of GLUT4 in fasting group was significantly lower than that in control group(P < 0.05).In myoblasts or SMSCs,insulin or glucose treatment at physiological concentrations up-regulated GLUT4 gene expression.Serum starvation for 18 h could significantly increase the expressorion of GLUT4 in myoblasts(P < 0.05).Experiment 3 Functional identification of chicken GLUT4 main transcriptsin skeletal muscle cellsTo explore the effects of GLUT4 gene on proliferation,differentiation,apoptosis and glucose uptake of chicken SMSCs.In the experiment,chicken myoblasts or SMSCs were used as the research object.The expression of GLUT4 in the process of proliferation and differentiation was detected by RT-qPCR.The biological effects of GLUT4 on the proliferation,differentiation,apoptosis and glucose metabolism of myoblasts or SMSCs were analyzed by liposome transfection technique.Finally,RNAseq was performed to further explore the pathway of GLUT4 gene function.The results showed that the expression of GLUT4 in the proliferation and differentiation stages of chicken myoblasts and SMSCs was different,and the expression level in the differentiation stage was lower than that in the proliferation stage(P < 0.05).After overexpression of GLUT4,RT-qPCR,CCK-8,EDU assays showed that overexpression of GLUT4 could promote the proliferation and differentiation of chicken muscle cells and inhibit apoptosis.It was found that overexpression of GLUT4 could promote glucose uptake by glucose uptake test and glucose content test in cell culture medium.GLUT4 was overexpressed in chicken SMSCs for 48 h,and RNA-seq was performed on the overexpression group and the control group cells.A total of 302 differentially expressed genes were obtained,which were mainly enriched in GO terms such as mitochondria,respiratory chain,transmembrane transporter activity and calcium binding.KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in oxidative phosphorylation,ribosome,myocardial contraction and cytochrome P450 metabolism of xenobiotics,indicating that GLUT4 may promote cell proliferation and differentiation and inhibit apoptosis by regulating mitochondrial-related genes to regulate cell glucose uptake. |
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