Effect Of Gastrin On The Secretion Of Insulin And Growth Hormone And Its Expression In Ovine And Bovine Tissue

Gastrin(GAS)is secreted by the antral sinus cells and released into the blood,mainly to promote the secretion of gastric juice.Gastrin and cholecystokinin(CCK)belong to the same family.The role of the GAS is mediated through its specific GAS/CCK-B receptor(named CCK-B).Previous study by our team showed that GAS has an effect on growth hormone(GH)and insulin secretion,but its possible mechanism is still unclear.In this study,bioinformatic analysis of amino acid sequence of GAS family and its receptors,the expression or localization of genes and protein in various tissues of sheep,injection assays in vivo and pituitary cell culture assays in vitro were explored for the possible mechanisms by which GAS regulates GH and insulin secretion.1.The difference between the amino acid sequence of GAS and CCK hormone and their receptors in different animal species was compared by UniProt database,and the whole amino acid sequence phylogenetic tree analysis was carried out.The results of the analysis showed that the C-terminal amino acid sequence of GAS and CCK were highly conserved among animals,while the N-terminus was a variable region.Phylogenetic analysis showed that CCK-A and CCK-B of sheep and cattle,rat and mouse,dog and cat were on the same branch;full amino acid sequence from GAS and CCK and their receptors(including signal peptides and originals),the sequence both avian and fish are larger than the mammals.the similarity of amino acid sequence between CCK-A and CCK-B receptors in sheep is only 45.094%.2.The intravenous injection test was performed using an incomplete Latin square test.Xinjiang fine wool sheep were tested for plasma insulin and blood glucose levels before and after a bolus intravenous injection of 20 nmol/kg BW GAS,GRP or 0.1%BSA saline(control group).The results showed that plasma insulin levels were not changed after GAS and GRP injection compared with control group(P>0.05).Glucose level after GAS injection 30 min was significantly lower than that of control group(P<0.05).Glucose level after GRP injection 10 min was significantly higher than that of control group(P<0.05).3.In order to explore the distribution of GAS and its CCK-B receptors in ovine tissue,the relative expression levels of GAS and CCK-B genes in four Xinjiang fine wool sheep were detected by real-time fluorescent quantitative PCR.The results showed that GAS gene expression was highest in the antrum of abomasum and was significantly higher than other tissue(P<0.01).CCK-B gene was highest in the abdomen and was significantly higher than other tissue(P<0.01),followed by in pituitary.Immunohistochemical localization showed that GAS immunopositive cells were only distributed in the mucosal layer of the antrum of abomasum,but not in the pituitary,atrophic fundus,kidney,testis and pancreas.4.In order to explore whether GAS promotes GH secretion directly through the pituitary to promote GH secretion,effect of 1 × 10-7mol/L GAS,GHRH(positive control)and 1 × 10-9 mol/L GAS on GH secretion in primary pituitary cells from 4 bulls was tested.Western blot results showed that GHRH positive control significantly increased GH levels in pituitary cells(P<0.01),but neither concentration of GAS affected GH level of supernatant(P>0.05).The above results indicate that the C-terminus of the GAS amino acid sequence is a conserved sequence and a biologically active site;GAS may be involved in the regulation of blood glucose levels in ruminants.Although the specific CCK-B receptor of GAS is expressed in the pituitary,the level of GH increase induced by GAS may not be directly affected by the pituitary.