| Obestatin is a recently identified 23-amino acid gut-derived peptide encodedby the preproghrelin gene. Although originating from the same precursor as the widely studied appetite-stimulating peptide ghrelin, obestatin was reported to bring on antithetical physiological effects with regards to food intake and body weight regulation. Obestatin and ghrelin were called yin and yang peptidefor encoded by the same gene and have opposite physiological functions in several systems. ghrelin, a 28-amino acid and ligand for the growth hormone secretagogue receptor-1a, has demonstrated to be a orexigenic peptide. As a ghrelin-associated peptide, obestatin was derived from the same proghrelin as ghrelin by different post-translational cleavage and borned with contestation. In 2005, zhang et al first identified obestatin as a 23-amino acid isolated from stomach mucosa and encoded by preproghrelin gene. The initial work showed that intraperitoneal or intracerebroventricular injection of human obestatin supressed food intake and decreased body-weight gain. Further studies indicated thatobestatin was involved in inhibiting thirst and anxiety , improving memory, affecting cell proliferation, controlling fluid homeostasis. However, controversialresults focus on its anorexigenic actions. In vivo and vitro studies, several investigators reported anorexigenic effects of peripheral obestatin in rodents. Furthermore, decreased gastric body mucosa obestatin expression and obestatin levels in plasma in obese patents indicated that obestatin may play an important role in the regulation of energy balance, apparently being able to antagonize the effects of ghrelin. In spite of those evidence, the anorexigenic effects of obestatin were not reproduced by subsequent studies using differernt dose of obestatin, experimental protocols and species or origin of the peptide. Different from ghrelin, obestatin has high affinity for GPR39 which belongs to the ghrelin receptor subfamily and hypothesized to be a congate ligand for GPR39. Binding studies and molecular approaches indicated that GPR39 was expressed ina wide range of tissues including central nevous system, peripheral organs, especially at a higher level in digestive system. Opposite to the initial report, later studies from different groups were unalble to corroboratre the binding between obestatin and GPR39. In 2007, Zhang et al. proved again that their original result of obestatin for GPR39 was unreproductive. Other receptor of obestatin was reported by Yasuda et al that Zn2+ could be a physiologically relevant agonist or modulator of GPR39.Pancreatic exocrine secretion is known to be modulated by a number of brain-gut peptides directly or through brain-pancreas anxis. It has been reported that cholecystokinin(CCK), secretin , pancreatic polypeptide(PP), ghrelin and amphetamine-regulated transcript peptide(CART) stimulate pancreatic secretion, whereas neuropeptide Y and somatostatin serve as inhibitors. Interestingly, like ghrelin, obestatin was found to stimulate the secretation of pancreatic juice enzymes through a vagal pathway in anaesthetized rats. Saleem and co-workers reported that obestatin inhibited the secretion of both somatostatin and PP in ratislets. These results need a further insight into the influence of obestatin on pancreatic secretion. Since previous study has investigated the inhibition effectsof exogenous ghrelin on pancreatic secretion through a vagal pathway. However,effect of exogenous obestatin on exotrine of pancreas await further confirmation. Therefore, the aim of the present study was to analyse the effect of amidated obestatin in rats on pancreatic secretion. CCK-8 was used as a positivecontrol for stimulation of pancreatic secretion. In order to verify the internalvalidity of our study design. Inaddition, the in vitro effects of obestatin was determined.FirsFirst part: A simple modified method for isolating and purifying the ratpancreatic acinar cellsObjective:To develop a simple and economical modified method for isolatingand purifying pancreatic acinar cells in rat．Methods:SD rats were starved for 12h with water given. Male SD rats(8-10weeks) weighting 200-250g were anesthetized by intraperitoneally injection ofsodium 3% pentobarbital and fixed on the operating plat. After sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleen was moved out. The pancreatic tissues were washedwith Hepes after intraabdominal anesthesia, then the pancreas is trimmed freeof fat and mesentery at room temperature(23℃), and 5ml of the collagenase(0.3 mg per ml in Krebs-Ringer bicarbonate solution) is injected into the interstitium of the tissue so as to distend the gland and rapidly expose the majority of its lobules to the enzymes. After 5 minutes, the tissue is cutted into small pieces about 2-4mm. The gland and excess enzyme solution is transferred to a 10ml erlenmeyer flask, equilibrated with 95%02-5% C02, and incubated at 37℃for 50 min with agitation at 60 oscillations per min. After the tissue structure collapse and loosens. Acinar cells are lightly liberated by sequential passage through pipets with tip diameters of 1.3 mm and 0.9 mm (5 times in each). The digestion was stopped by Krebs-Ringer bicarbonate solution containing 4% bovine-plasma albumin. Finally,acinar cells were harvested in conical centrifuge tubes, and centrifuged at 50g for 5 min to form pellets of packed cells and places them in a protective environment. After two further washes ofthe pooled cells in 8 ml of the above solution, they are suspended in Krebs-Ringer bicarbonate buffer containing 1% bovine-plasma albumin. All subsequent incubations are performed at 37℃.ResultsResults: The cell count(5-6×106)and purification (70±6)％of the pancreatic acinar cels harvested by the modified method had no statistical diference compared with Wang's method. The cell activity (>90％)of modified method group was higher than the control method group．Conclusion:Modified method is a simple and effective method for isolating and purifying the pancreatic acinar cells．Second part: Effect of different doses of obestatin on amylase of pancreatic acinar/lobules in ratsObjective: To investigate the effects of different doses of obestatin on exocrine of pancreatic acinar/lobules in rats.Methods: Preparation of dispersed pancreatic acini was performing according to improved method of Williams et al. The isolated pancreatic acini were incubated with different doses of obeatatin(0.1,1,10,30nmol/L)with or without CCK-8(10-10mol/L)for 1 hour. The acinar cells contained within the pellet were lysed with a sonicator for 5 minutes. Amylase content in the supernatants and pellet was detected using the Phadebas reagent and expressed as asa percentage of total amylase content. Pancreatic lobules, containing intrapancreatic nerve terminals and islets in addition to exocrine cells,were prepared bythe method of Scheele＆Palade. Male SD rats (8-10weeks) weighting 200-250gwere anesthetized by intraperitoneally injection of sodium 3% pentobarbital and fixed on the operating plat. Ater sterilzed in 75% alcohol for 30minutes, the rat was made a midline incision, pancreas between duodenum and spleenwas moved out. The pancreatic tissues were washed with Hepes after intraabdominal anesthesia, then the pancreas is trimmed free of fat and mesentery at r oom temperature(23℃),The pancreatic tissue was divided into 4mm×5mm lobular unites; Pancreatic lobules were incubated with different concentrations of obestatin for 30min at 37℃with or without KCL. The medium was then aspirated, diluted 1:10 and assayed for amylase.To the remaining tissue, 1ml KHB was added and the tissue was homogenized. Amylase in medium and tissue homogenates were assayed and expressed as the percentage of total tissue content.ResultResults: Graded contrations of obestatin alone produced no significant changein basal amylase release and has no dose-dependent manner(P >0.05). Co-incubation of CCK-8 with various concentrations of obestatin produced no alteration on amylase release compared to that observed with CCK-8 alone(P >0.05).Incubated with potassium significantly increased amylase relase in pancreatic lobules(P <0.05). Obeatatin produced no change on amylase release withpotassium in pancreatic lobules(P >0.05).Conclusions: Our results demonstrate that exogenous obestatin has no effectson pancreatic exocrine of rats in vitro study.Third part: Effect of intravenous injection of obestatin on pancreatic exocrine secretion in ratsObjective: To evaluated the effects of intravenous injection of obestatin on pancreatic exocrine secretion in rats.Methods: Sprague-Dawley weighting 250-300g rats were randomly divided into three groups(group A,B,C). The pancreatic exocrine model was made beforeexperiments. After anesthetized with intraperitoneal injection of 3%sodium pentobarbital. The rats were fixed on an operating plat and sterilzed by 75% alcohol for 30minutes,then the rat was made a midline incision,Two polyethylenecatheter (PE-10) were respectively placed into the femoral vein and tail veinfor infusion of obestatin and other drugs using a syringe-driven pump. The third polyethylene cannula (PE-10) was inserted into the common bile-pancreatic duct at Oddi's sphincter. To permit the return of pancreatic-bile juice (P-BJ),the forth cannula was advanced into the duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volumewas measured, and an aliquot was taken and diluted with distilled water for protein determination.The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. Protein i n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B,two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.Forth part Effect of central administration of obestatin on pancreatic secretionObjective:The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .Methods:Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi's sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest n the P-BJ was measured spectro-photometrically by a BCA protein assay. Ingroup A and B,two obestatin boluses (1nmol kg-1h-1 and 5nmol kg-1h-1) weregiven intravenously. After 45-min, CCK-8 were given from femoral vein at adose of 400pmol kg-1h-1. In group C, CCK-8 was given alone as control.ResultsResults: Intravenous infusion of CCK-8 evoked a significant increase in P-BJand pancreatic protein secretion (from 23.7±2.5 to 47.6±2.0 mg/15 min, P< 0.05). Contrast to the baseline levels, the volume of P-BJ and the protein output have no signifigant change in group A and B(15min, 30min; P > 0.05).The signifigant increase of protein secretion stimulated by CCK-8 was not change after injection of obestatin.Conclusions: Intravenous injection of obestatin has no impacts on the volumeof P-BJ and the protein output. Combined injection of obestatin did not affect CCK-8–induced pancreatic secretion.Forth part Effect of central administration of obestatin on pancreatic secretionObjective:The present study was set up to bring more clarity into the contested effects of central administration of obestatin on pancreatic secretion .Methods:Sprague-Dawley rats were randomly divided into three groups (group A,B,C). The pancreatic exocrine model was made before experiments. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital. Apolyethylene catheter (PE-10) was placed into the tail vein for infusion of CCK using a syringe-driven pump. The second polyethylene cannula (PE-10) wasinserted into the common bile-pancreatic duct at Oddi's sphincter. To permitthe return of pancreatic-bile juice (P-BJ), the forth cannula was advanced intothe duodenum slightly above the ampulla. Combined P-BJ was collected every 15 minutes after 30-min. The volume was measured, and an aliquot was taken and diluted with distilled water for protein determination. Protein in the PBJwas measured spectro-photometrically by a BCA protein assay. The remainder of the undiluted P-BJ was pumped back into the rat via the duodenal cannula during the next collection period. After that, an incision was made in thescalp, and a small hole was drilled in the skull using a dental drill. A micropipettes was positioned 1.0 mm lateral and 0.46 mm posterior to bregma, and2.20mm ventral from the skull surface. After surgery, animals were allowed to recover 30min prior to experiment. The micropipettes were filled with obest period. In group B, intestinal perfusion of obestatin was performed at a speedof 2ml/h for 105min. The control group had no intestinal infusion.ResultsResults: After the intraduodenal infusion of obestatin, the volume of combinedbile-pancreatic secretions and the protein output was not changed contrast tocontrol group( P > 0.05).Conclusions: Intraduodenal infusion of obestatin has no effects on the volumeand the protein output of combined bile-pancreatic secretions... |
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