| Dietary protein and amino acids are important source of nutrtion and an important signaling molecules which regulate a variety of physiological processes in the gastrointestinal tract,including enteric nervous system excitability,proliferation and differentiation of intestinal epithelial cells,secretion of gastrointestinal hormones,subsequently regulate the physiological functions that include the motility of gastrointestinal tract,digestion and absorption of nutrients.Recent reseach suggested that calcium sensing receptor(CaSR)was activated by protein and amino acids in gastrointestinal endocrine cells,increased intracellular Ca2+ mobilization,promoted production and secretion of gastrointestinal hormone.The studies that amino acids and protein impacted the expression and functions of CaSR in gastrointestinal tract mainly focused on human,rat and mouse.However,whether CaSR expressed in the gut of porcine and whether the receptor expression was regulated by protein and amino acids remain unknown.Recent studies indicated that the aromatic amino acids,especially L-phenylananine(Phe)and L-tryptophan(Trp)could stimulate the secretion of gastrointestinal hormones in STC-1 cells and small intestine of mouse,which may be mediated by the CaSR.Whether Phe and Trp stimulated secretion of gastrointestinal hormone in small intestine of pig and whether CaSR mediated this effect are need to calrify.This thesis is composed of the following parts:1 Effects of dietary protein content on CaSR gene expression in small intestine and gastrointestinal hormone secretion in serumThe objective of this experiment is to investigate the effect of dietary protein content on the expression of CaSR gene in small intestine and the secretion of serum gastrointestinal hormone in different physiological stage of pigs,and to explore the relationship between CaSR expression and gastrointestinal hormone secretion.Total 54 cross-bred(Duroc × Landrace × Yorkshire)pigs with good health and similar body weight were selected,including 18 weaned pigs at 10kg(9.57 ± 0.64kg),18 growing pigs at 30 kg(36.47 ± 0.20 kg)and 18 finishing pigs at 60 kg(62.30 ± 0.07 kg),respectively.Eighteen pigs in different stage were randomly divided into 3 groups and fed with corn-soybean meal basal diet.The dietary crude protein(CP)level for weaned pigs were 20%(NRC standard),17%14%,those for growing pigs were 18%(NRC standard),15%,12%,and those for finishing pigs were 16%(NRC standard),13%,10%.After 45 days experimental period for weaned pigs,30 days for growing pigs and 50 days for finishing pigs,respectively,all pigs were slaughtered.The precaval vein blood and the small intestine tissue were collected for the analysis of gastrointestinal hormones concentration and CaSR expression.With the reduction of dietary protein,the concentration of CCK,GLP-1 and PYY in serum showed a significant decrease in piglets,and the same alteration as the CCK,GIP concentrations in growing pigs and GIP concentration in finishing pigs(P<0.05).Low protein diets significantly decreased the expression of CaSR in small intestine among piglets,growing and finishing pigs(P<0.05).The correlation analysis suggested that a significant positive correlation were revealed in the expression of jejunal and ileal CaS/R and the concentration of GLP-1 and GIP,the same variation between the expression of ileal CaSR and the concentration of PYY in piglets.In growing pigs,there was a significant positive correlation between the expression of CaSR in duodenum and the concentration of CCK,and the expression of jejunal and ileal CaSR and the concentration of GIP also showed a a significant positive correlation.Our findings suggested that the reduction of dietary protein level by 3%or 6%decreased the expression of CaSR and the secretion of gastrointestinal hormones(e.g.CCK).The gastrointestinal hormone secretion may be mediated by CaSR,which needs further investigation to verify.2 Phe-induced secretion of gut hormone mediated by CaSR in duodenum of pigsPhe,one of the essential amino acids,regulated gastrointestinal hormone secretion by activating CaSR in small intestine of human and rodents.This experiment focused on the relation and regulation of CaSR-mediated gastrointestinal hormone secretion by phenylalanine and potential role of its downstream signaling pathways in duodenum of swine using superfusion system.The results showed that phenylalanine significantly promoted the secretion of CCK,GLP-1,GIP and PYY,increased the CaSR and gastrointestinal hormone gene expression(P<0.05)and the level of CaSR and its downstream signaling proteins(P<0.05).NPS 2143 is an antagonist of CaSR,which inhibited phenylalanine-induced the secretion of CCK,GLP-1 and GIP(P<0.05),down-regulated the expression of gastrointestinal hormones and CaSR gene(P<0.05),deceased phenylalanine-induced the level of CaSR and its downstream signaling proteins(P<0.05).Ca2+ is natural ligand of CaSR,which promoted the phenylalanine-induced the secretion of gastrointestinal hormone(P<0.05),increased the expression of CaSR and gastrointestinal hormone gene(P<0.05).In summary,phenylalanine has a role that can promote the secretion of gastrointestinal hormone in duodenum of porcine,which may be mediated by CaSR and subsequently initiated the G protein-coupled signal transduction through the activation of PKC,IP3R and PKA.3 Trp-induced secretion of gut hormone mediated by CaSR in duodenum of pigsSimilar to phenylalanine,tryptophan is also a crucial signal molecule,playing a regulatory role in a variety of physiological processes in the body.Recently,lots of studies found that tryptophan is the endogenous ligand of CaSR derived from nutrients,which activated CaSR and regulated a variety of physiological processes,including secretion of gastrointestinal hormones.This experiment mainly studied the relation and underlying mechanism that secretion of gastrointestinal hormone was regulated by tryptophan in porcine duodenum.It was found that tryptophan promoted duodenal CCK,GLP-1,GIP secretion(P<0.05),raised expression of CaSR,CCK,GIP and PYY gene(P<0.05)and the level of CaSR and its downstream signaling protein(P<0.05).NPS 2143 is an antagonist of CaSR,which inhibited tryptophan-induced secretion of CCK,GLP-1,GIP,and down-regulated expression of tryptophan-induced CaSR and CCK,GIP,PYY gene(P<0.05),inhibited the level of CaSR and its downstream signaling proteins(P<0.05).Ca2+ is the natural ligand of CaSR,which significantly facilitated secretion of gastrointestinal hormone that caused tryptophan(P<0.05),raised the expression CaSR,CCK,GIP gene that induced by tryptophan(P<0.05).The results indicated that tryptophan promoted the secretion of gastrointestinal hormones in pig duodenum,the mechanism of this effect may be mediated by CaSR,subsequently initiated the G-protein coupled downstream signal transduction through the activation of PKA,PKC,IP3R,then the intracellular Ca2+raised.Thereby,the gastrointestinal hormone was secreted from duodenum of pigs. |
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