Research On The Effects And Pathways Of Amino Acids Regulating Insulin Secretion

Previous studies suggested that endocrine may be one of the pathways by which AA supply affecting milk protein synthesis.Insulin,as the sole hormone that reduces plasma glucose concentration,has been reported to play big roles on milk protein production.However,the effects and pathways of AA regulating insulin secretion in lactating animals need to be systematically investigated.The objective of this study was to observe the effects of different EAA on insulin secretion in lactating rats,mice and goats as well as?cell lines,and hope to give some reference for the evaluation of AA requirement.1.Effects of dietary protein level on insulin concentrations in lactating rats.Eighteen lactating rats were assigned either to 9%,21%and 35%protein diets for 15 d at delivery to investigate the effects of dietary protein level on insulin and glucagon concentrations in plasma and pancreas.The 35%protein diet increased the circulating insulin and glucagon levels than the 9%protein?25%and 17%?and 21%protein?37%and 31%?diets;while their pancreatic contents in 9%protein and 35%protein groups were lower than the 21%one.Hepatic PEPCK and G6Pase activities in 35%protein group were greater than those in the 21%and 9%protein groups.When combined the three groups,the plasma NO concentrations were correlated negatively to circulating insulin concentrations,and were positively to pancreatic insulin,glucagon,and IGR levels.Results of regression analysis revealed that the circulating insulin levels were mainly correlated to plasma concentrations of Thr and Phe,and the circulating glucagon levels were mainly correlated to concentrations of Val,Ser,Asp,and Cit.2.Effects of graded EAA supply on insulin secretion and mitochondrial metabolism in?cell of rat.INS-1 cell were cultured for 24-h in medium containing 0%,20%,40%,60%,80%and100%of total EAA to investigate the effect of EAA supply on insulin secretion and mitochondrial metabolism.Graded increase in EAA concentration of medium decreased the insulin release,intracellular insulin content and gene expressions of AA transport quadratically,but had no effects on Ins2 mRNA abundance in INS-1 cell.On the other hand,graded increase in EAA concentration changed the intracellular ATP content quadratically and had a linear decrease in the gene expressions of Tfam,Ucp2 and mitochondrial key enzymies,though the glucose uptake and glucose transport gene expression were not affected by the treatments.3.Effects of single EAA stimulation on insulin secretion and mitochondrial metabolism in?cell of rat.INS-1 cell were cultured in KRBH with 11.1 mM glucose and 10 mM single EAA for 1h to investigate the effect of single EAA on acute insulin secretion and mitochondrial metabolism.Compared with the treatment without EAA,high Arg concentration increased the insulin secretions of INS-1 cell,while the Trp administration reduced insulin secretions.However,these two treatments significantly decreased the intracellular ATP contents.Real time-PCR analysis revealed that His,Leu and Thr treatments decreased the Tfam gene expressions,and His,Ile and Thr treatments increased the Pdx1 gene expressions relative to the None treatments.The Nd4l mRNA abundance in Ile and Phe groups were lower,and the Odgh gene expressions were higher than those in the None groups.In addition,supplementation of Arg to the None medium significantly elevated the intracellular Ca²⁺concentration.4.Effects of Arg supplementation or deficiency on insulin concentrations in lactating mice.Thirty-two lactating mice were assigned to 9%protein diet?LP?,9%protein+arginine diet?LP+Arg?,21%protein diet?HP?or 21%protein-arginine diet?HP-Arg?for 15 d at delivery to investigate the effects of Arg supplementation or deficiency on insulin secretion,hepatic gluconeogenesis and Arg metabolism.The amounts of supplementation or deficiency were equal to the Arg content in 12%protein diet.Compared with the LP diet,HP diet increased the weight gains of dams and pups?6/dam?as well as plasma glucose,NO and Arg concentrations.Arg supplementation to the LP diet increased the plasma insulin concentrations and IGR,but decreased the glucagon contents in pancreas.What's more,Arg deficiency from the HP diet elevated the circulating insulin levels and IGR,and reduced the pancreatic insulin and glucagon concentrations.On the other hand,both supplementation and deficiency of Arg decreased the hepatic PEPCK,G6Pase and Arginase activities.In addition,LP+Arg treatment decreased the activities of RPMT than the LP treatment,but the NOS activities of lactating mice in HP-Arg group were higher than those in the HP group.5.Effects of different Arg supply on insulin secretion and mitochondrial metabolism in?cell of mouse.MIN6 cell was cultured in medium containing 0,0.04,0.4?control?,4 and 8 mM Arg for24 h to investigate the Arg supply on insulin secretion and mitochondrial metabolism.Both supplementation or deficiency of Arg from the basal medium decreased the cell activities,but increased the NOS gene expression and PKC activities.Besides,compared with the 0.4 mM treatment,0.04 mM treatment increased the insulin secretion,but 4 and 8 mM treatments decreased the insulin secretion.Besides,0 and 0.04 mM treatments elevated the intracellular insulin contents and GCK activities,but reduced the uptakes of glucose by MIN6 cell than the control treatment.6.Effects of different jugular AA infusion mixtures on insulin concentrations in lactating goats.Five lactating goats were fasted for 24 h followed by intravenous infusions of a mixture containing AA and glucose for 8 h to investigate the effects of ILMT and RAA on islet hormone concentrations and lactational performance.Treatments were containing no total AA?NTAA?,3×the MR of ILMT?3F0R?,and 3F0R plus 1×,2×,or 3×MR of RAA?3F1R,3F2R and 3F3R,respectively?according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat.Relative to NTAA,supplementation of ILMT tended to increase the milk protein production and plasma glucose concentrations,and increased milk and lactose production,but had no effects on the production or contents of milk fat.Graded supplementation of RAA tended to quadratically affect production of milk and lactose.Arterial glucose and glucagon concentrations decreased linearly,and plasma insulin concentrations decreased quadratically with increased RAA.Mammary P70S6K phosphorylation was decreased by addition of ILMT as compared to NTAA,but increased linearly with increased RAA infusion.These results suggested that:?1?Variations in circulating AA and NO concentrations and hepatic gluconeogenic enzyme activities are likely intermediary responses involved in the effects of dietary protein levels on synthesis and secretion of islet hormones in lactating rats;?2?Graded increase in EAA concentration of medium decreased the insulin secretions and intracellular insulin contents quadratically,which accompanied with the linearly decreased gene expressions of key enzyme in mitochondrial metabolism and regulated factors,suggesting the big roles of mitochondrial metabolism in the EAA supply induced variations in insulin secretion.?3?High concentration of Arg induced a initial increase of Ca²⁺concentration and promoted the acute insulin secretion in INS-1 cell,while Trp had a depressed effect on acute insulin secretion,but the other EAAs had no effects on insulin secretion.?4?AA imbalances induced by the supplementation or deficiency of Arg from the diet and medium have effects on the insulin secretion and glucose uptake by?cell.?6?ILMT and RAA had different effects on insulin,and divergent responses to these 2 groups of AA may occur duo to the AA concentrations in lactating goats.