The Effects And Molecular Mechanisms Of Orexin A And OX₁R On The Function Of Pancreatic β Cells In Obese IR Rats

Preface:With the changes of life style,the incidences of metabolic disorders increased year by year,such as diabetes,obesity,and so on.Those diseases have a common pathogenesis:insulin resistance（IR）.Therefore,there are numerous studies focus on IR.The recent opinion on the reason of IR is the deficiency of the transduction pathway of insulin signaling.The recent studies suggest that the transduction pathway of insulin signaling existed in the pancreaticβcells.The deficiency of the transduction pathway inβcells may resulting in the disturbance of those function.Orexin A and orexin B（also known as hypocretin-1 and hypocretin-2）are a group of neuropeptides,which were originallly discovered in rat hypothalamus in 1998.They proteolytically processed from a common precursor preproorexin.Collectively,orexins play roles in many physiological functions,including arousal,sleep-awake,feeding behavior,reproductive behavior,stress reactions,energy homeostasis and glucose metabolism.Orexins act through two subtypes of G protein-coupled receptors,orexin receptor-1（OX₁R）and orexin receptor-2（OX₂R）.OX₁R was the first of the two orexin receptors to be discovered and has 10-fold higher affinity with orexin-A compared with orexin B,whileOX₂R was concluded to be a nonselective receptor.Orexin A and its receptors are widely dispersed throughout the central nervous system and periphery tissues,including neuronal and endocrine cells of the gastrointestinal tract,adrenal gland and pituitary.Several studies demonstrated the expression of OX₁R in the endocrine pancreas in humans and rodents,suggesting that the effects of orexin A in this tissue are mediated through a direct interaction with OX₁R.Additionally,orexin A and OX₁R are involved in the regulation of cell proliferation and apoptosis.Despite the important biological action of orexin A,to date,the studies characterizing the direct effects of orexin A on the function of pancreaticβcells were inconsistent,and the mechanism was still not clear.Therefore,in this report,we observed the expression of OXA and OX₁R in high-fat-diet induced obese rats,the changes of insulin signaling pathway in those rats,investigated the role of orexin A in the regulation of insulin secretion and cell proliferation and apoptosis of pancreaticβcells,and characerized the underlying mechanisms of OX₁R evoked insulin receptor signaling pathway involved in this process.Methods:1.The expression of orexin A and OX₁R in high-fat-diet induced IR ratsForty wistar rats were randomly divided into two groups:normal control（NC）,high-fat-diet group（HF）,and fed with normal diet or high-fat-diet,respectively.At the end of eight weeks,the glucose infusion rate（GIR）was measured by using euglycemic hyperinsulinemia clamp to evaluated the peripheral insulin resistance.We determined the levels of serum fasting blood glucose（FBG）,fasting insulin（FIN）,triglyceride（TG）and the levels of plama orexin A.The rats in the two groups were sacrificed,and the pancreatic islets were isolated and collected.The expression of insulin receptor substrate-1（IRS-1）,insulin receptor substrate-2（IRS-2）,phosphatidylinositol-3-kinase（PI3K）,protein kinase B（AKT）mRNA were detected by RT-PCR.The expression of OX₁R were detected by immunohistochemistry and western blot.2.Effects of orexin A and OX₁R on the secretion function,cell proliferation and apoptosis of pancreaticβcellsINS-1 cells were treated with OXA of different concentrations((10^-1010 M,10^-8M,10^-6M)and/or SB334867（OX1R antagonist）and/or LY294002（PI3K antagonist）.We determine the insulin secretion of the INS-1 exposure to normal glucose and high concentration glucose used RIA essay.Then,we detected the expression of OX₁R by western blot,detected cells proliferation by MTT,and cells apoptosis by ELISA rit.3.Effects of orexin A and OX₁R on the insulin signaling transduction pathway of pancreaticβcellsINS-1 cells were treated with OXA of 10^-88 M and/or SB334867（OX1R antagonist）.We detected the total protein and phosphorylation protein expression of insulin receptor substrate-1（IRS-1）,insulin receptor substrate-2（IRS-2）,phosphatidylinositol-3-kinase（PI3K）,protein kinase B（AKT）by western blot.Results:1.The expression of orexin A and OX₁R in high-fat-diet induced IR rats（1）The body weight and visceral fat mass of the rats in HF group were dramatically higher than in NC group.（2）The TG concentration of blood in HF group were higher than in NC group.The FBG levels of IR rats in HF group was higher than in NC group,but not statistically.（3）The FINs of blood in HF group was higher than in NC group,but GIR was lower than in NC group.（4）RT-PCR results showed that the expression of IRS-1,IRS-2,PI3K,AKT mRNA in the pancreatic islets of HF group were lower than in NC group.（5）Immunohistochemistry results showed that the expression of OX₁R in HF group was lower than in NC group.（6）ELISA results showed that the levels of plama orexin A in HF group was higher than in NC group.2.Effects of orexin A and OX₁R on the secretion function,cell proliferation and apoptosis of pancreaticβcells（1）Orexin A induced the expression of OX₁R in INS-1cell.The concentra-tion of10^-10M and 10^-8M had statistical effect.（2）Orexin A promoted the insulin secretion of INS-1cell.The concentration of10^-8M had the optimal effect.When pretreated with SB334867,the secretion was reversed.LY 294002 could partly reverse the effect.（3）Orexin A promoted the cell proliferation of INS-1cell.The concentration of10^-8M and 10^-10M had statistical effect.When pretreated with SB334867,the effect was totally reversed.LY 294002 could partly reverse the effect.（4）Orexin A also decreased the apoptosis of INS-1cell.The concentration of 10^-8M and 10^-10M had statistical effect.When pretreated with SB334867,the effect was totally reversed.LY 294002 could partly reverse the effect.3.Effects of orexin A and OX₁R on the insulin signaling transduction pathway of pancreaticβcells（1）Compared with normal group,exposure to 10^-8M orexin A significantly induced the phosphorylation of IRS-1,IRS-2,PI3K and AKT.And the total protein expression of IRS-1,IRS-2,PI3K and AKT were unchanged.（2）SB334867 could not increased the phosphorylation of IRS-1,IRS-2,PI3K and AKT alone,but it could reverse the effect of orexin A.4.Effects of orexin A and OX₁R on proliferation of PANC 1 cells.（1）Compared with protein expression of OX1R in PANC 1 cells and HPC-Y5 cells.（2）Orexin A stimulate proliferation of PANC 1 cells via OX₁R,SB408124 could reverse the effect of orexin A.Conclusion:1.High-fat-diet induced the occurring of IR rat models,which showed an impaired expression of insulin signaling transduction pathway.2.The level of plasma orexin A was higher,and the expression of OX₁R in pancreas was lower,which presents that they may correlated with the function of pancreaticβcells.3.Orexin A promoted the insulin secretion,cell proliferation and reduced the apoptosis of INS-1 cells by inducing the OX₁R and PI3K pathway.4.Orexin A up-regulated the insulin signaling pathway through OX₁R,and participated the regulation of INS-1 cells.5.OX₁R highly expressed in pancreas tumor cells,Orexin A stimulate proliferation of PANC 1 cells through OX₁R.