ghrelin , a 28-amino acid peptide , is produced mainly in the stomach and to a lesser extent in the small intestine , pancreas , and hypothalamus and is the endogenous ligand for the growth hormone secretagogue receptor ( ghs - r ) [ 19 ] . serine-3 of ghrelin is acylated with an octanoic acid , the process catalyzed by a recently discovered ghrelin o - acyltransferase ( goat ) [ 10 , 11 ] . the acylation is thought to be required for its biological activity , although desacyl ghrelin has been reported to exert several effects . administration of pharmacological doses of acylated ghrelin (= ghrelin ) to intact animals increases food intake , induces body weight gain , and causes obesity [ 1318 ] . the orexigenic and body fat promoting properties of ghrelin and growth hormone ( gh ) secretagogue ( ghs ) are thought to be independent of gh and mediated primarily by the hypothalamic neuropeptide y ( npy ) and agouti - related protein ( agrp ) systems [ 1416 , 1922 ] . although pharmacologic effects of ghrelin are well documented , physiological role of endogenous ghrelin is poorly understood . it has recently been reported that knockout of either the ghrelin gene or ghrelin receptor gene exerts no or minor effects on body weight and body composition [ 2325 ] . however , the lack of phenotypic changes in knockout mice might reflect compensatory mechanisms that are known to operate occasionally . in humans , gastrectomy results in loss of body weight of about 10% within the first six months after surgery , primarily due to reduced body fat . in addition , gastrectomized patients often complain of loss of appetite , general fatigue , and in some cases impaired bone quality such as osteopenia and osteomalacia [ 9 , 27 ] . at present there are no satisfactory mechanistic explanation and treatment for any of these symptoms . the effects of gastrectomy on food intake and body composition have been poorly documented in rodents . loss of ghrelin could be implicated in these symptoms , since as much as 80% of circulating ghrelin is lost following surgical removal of the glandular stomach or the acid producing part of the stomach in rats and humans [ 4 , 28 , 29 ] . in this study , we performed total gastrectomy in rats and examined whether the recovery from gastrectomy - associated anorexia depends on the circulating ghrelin level or other factors and studied temporal changes in the biosynthesis and orexigenic ability of ghrelin following gastrectomy . male 4-week - old wistar rats ( slc , japan ) were maintained on a 12-hour light / dark cycle and given conventional food and water for 2 weeks and not deprived of food before gastrectomy . they were operated at 6 weeks of age with body weight around 130180  g. rats were anesthetized with an intraperitoneal injection of pentobarbital ( 40  mg / kg ) and a median abdominal incision was made . after the stomach was separated from the greater and lesser omentum , the duodenal bulb was ligated and transected . after the left gastric artery was ligated , the esophagus was clamped above the esophagogastric junction and the stomach was resected . an end - to - side anastomosis between esophagus and jejunum at the 4 - 5  cm anal side from treitz ligament was performed with 70 monofilament polyglyconate synthetic absorbable string ( maxon ; johnson & johnson inc . usa ) using interrupted suture ( billroth ii ( b - ii ) reconstruction ) . on the other hand , jejunum was transected at the 2 - 3  cm anal side from the treitz ligament . then , an end - to - side anastomosis between the esophagus and jejunum was performed with 70 maxon using an interrupted suture . jejunojejuno anastomosis was also done by end - to - side method ( roux - en - y ( r - y ) reconstruction ) . the abdominal wall and skin closure was made by 50 nylon ( johnson & johnson inc . after the operation , the rats were allowed only clear water without food for 3 days , dry milk from the 3rd day , and from the 7th day a conventional pellet diet with free access to water . after the operation , food intake for 24 hours and body weight were measured once a week for 3 months . the rats were sacrificed at three months after operation . to measure plasma ghrelin concentrations , duodenum samples of about 3  cm were taken from 1  cm anal side of duodenal stump . jejunum samples of about 3  cm were taken from 1  cm anal side of esophagojejunostomy . one antiserum was raised against the cooh - terminally cys - extended rat ghrelin ( position 1 - 11 ) in new zealand white rabbits ( # g606 ) that was shown to specifically recognize ghrelin with n - octanoylated ser 3 ( acylated ghrelin ; ghrelin ) . by the radioimmunoassay ( ria ) using this antiserum , designated nh2-terminal ria ( n - ria ) , the concentration of acylated ghrelin was obtained . the other antiserum was raised against the nh2-terminally cys - extended rat ghrelin ( position 13 - 28 ) ( # g107 ) that was shown to recognize both acylated ghrelin and desacyl ghrelin . by the ria using this antiserum , named as cooh - terminal ria ( c - ria ) these two ghrelin - specific rias were used to measure acylated ghrelin and desacyl ghrelin contents in several tissues . the bound and free ligands were separated using a second antibody . acylated ghrelin  is expressed as  ghrelin  and  acylated ghrelin plus desacyl ghrelin  is expressed as plasma concentrations of acylated ghrelin and desacyl ghrelin were measured using elisa kits ( mitsubishi kagaku iatron , tokyo , japan ) . ghrelin ( peptide institute , osaka , japan ) or saline was administered subcutaneously to control normal rats ( eight - weeks - old ) and those received gastrectomy with b - ii reconstruction . for ghrelin treatment at 2 and 6 weeks after gastrectomy control and gastrectomized rats were divided into two groups ; one group was treated with ghrelin and the other with saline , and food intake and body weight were measured . the injection of ghrelin ( 10  nmol / kg body weight ) was carried out once a day for seven days , and food intake and body weight were measured . for ghrelin treatment at 12 weeks after gastrectomy , the following procedures were used . single injection of ghrelin or saline was followed by measurements of food intake for the subsequent 24 hours . in the next week , two groups were changed : one group that had first received ghrelin was injected with saline , and the other group that had first received saline was injected with ghrelin . food intake after the first injection and that after the second injection were pooled and averaged . statistical analysis was performed using student 's paired and unpaired t - tests . values of p < .05 were considered statistically significant . food intake for 24 hours and body weight were measured in total gastrectomized and control rats during 12 postoperative weeks . gastrectomy with b - ii ( n = 40 ) and r - y reconstruction methods ( n = 35 ) both markedly decreased food intake . in both b - ii and r - y groups , at the 1st week after operation food eaten for 24 hours decreased to a level around 50% of that in control rats ( figure 1(a ) ) . in b - in contrast , food intake in r - y group increased gradually during 4 postoperative weeks . the daily food intake averaged for postoperative 12 weeks was significantly ( p < .05 ) reduced in b - ii and r - y groups ( figure 1(a ) ) . food intake in r - y group was significantly ( p < .05 ) greater than in b - ii group throughout postoperative 12 weeks . body weight significantly decreased after gastrectomy in both b - ii and r - y groups ( figure 1(b ) ) . body weight in r - y group was significantly greater than that in b - ii group from the 2nd through 12th postoperative week ( p < .05 ) . the correlation between body weight , 24 hours food intake , and plasma ghrelin concentrations at the 12th postoperative week was studied . food intake and body weight were correlated with each other irrespective of reconstruction methods and were plotted on a single regression line ( figure 2(a ) ) . in contrast , there was no correlation between food intake and plasma concentrations of total ghrelin ( figure 2(b ) ) . we next examined whether body weight correlates with plasma concentrations of ghrelin or total ghrelin , by using n - ria and c - ria . body weight did not correlate with plasma ghrelin and total ghrelin levels ( figures 2(c ) and 2(d ) ) . furthermore , no significant difference between r - y and b - ii groups was observed in averaged ghrelin levels ( b - ii : 46.3  7.2  fmol / ml ( n = 40 ) , r - y : 35.9  4.6  fmol / ml ( n = 35 ) ) and total ghrelin levels ( b - ii : 393.0  40.0  fmol / ml ( n = 40 ) , r - y : 349.8  23.2  fmol / ml ( n = 35 ) ) , though some rats in b - ii groups showed higher levels of ghrelin and total ghrelin . these results indicate that plasma ghrelin levels are significantly reduced in r - y and b - ii groups and that greater food intake in r - y than in b - ii is related to the method of reconstruction but not plasma ghrelin levels . plasma ghrelin concentrations were markedly reduced in both r - y and b - ii groups to the levels of about 30% of those in control rats ( figure 3(a ) ) . therefore , the reduction of plasma ghrelin levels is due to lack of release of ghrelin from stomach . we examined whether the reduction of ghrelin due to gastrectomy could influence the production of ghrelin in the intestine and pancreas , the tissues known to produce ghrelin . ghrelin concentrations in the duodenum increased approximately by 100% in r - y and b - ii groups ( figure 3(b ) ) , while those in the jejunum did not change ( figure 3(c ) ) . ghrelin concentrations in the pancreas were much smaller than those in the intestine , however , they dramatically increased approximately by 500% in b - ii and by 400% in r - y groups ( figure 3(d ) ) . on the other hand , plasma concentrations of total ghrelin were approximately five times higher than those of ghrelin , and they were markedly reduced by gastrectomy in both groups ( figure 3(e ) ) . contents of total ghrelin in the duodenum and pancreas , but not jejunum , increased in both r - y and b - ii groups ( figures 3(f)3(h ) ) . the gastrectomy - associated relative changes of total ghrelin in the circulation , duodenum and pancreas were similar to those of ghrelin . these results suggest that gastrectomy - induced reductions in circulating ghrelin levels promote synthesis of ghrelin in the duodenum and pancreas , which may have compensatory roles including the restoration of circulating ghrelin levels . in consistence with this inference , plasma levels of ghrelin and total ghrelin that were markedly reduced at 2 weeks after gastrectomy with b - ii reconstruction ( ghrelin : 25.0  5.3  fmol / ml ( n = 4 ) , total ghrelin : 295.8  34.6  fmol / ml ( n = 4 ) ) were significantly ( p < .05 ) elevated at the 12th postgastrectomy week ( ghrelin : 46.3  7.1  fmol / ml ( n = 40 ) , total ghrelin : 393.0  40.0  fmol / ml ( n = 40 ) ) . we examined whether reduced levels of plasma ghrelin after gastrectomy could be altered by fasting , the physiological regulator of ghrelin release . at the 12th postoperative week , blood samples were taken from ad - lib fed and 48 hour fasting rats . effects of fasting on plasma levels of ghrelin and desacyl ghrelin were examined by using elisa , since ria did not allow us to determine exact levels of desacyl ghrelin . .05 ) elevated by fasting approximately by 3 times in both b - ii and r - y groups ( figures 4(a ) and 4(c ) ) . desacyl ghrelin concentrations were also significantly ( p < .05 ) elevated by 3 - 4 times by fasting in b - ii and r - y groups ( figures 4(b ) and 4(d ) ) . thus , fasting induced similar fold increases in both ghrelin and desacyl ghrelin levels in the circulation in gastrectomized rats . rats aged 8-weeks were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for seven continuous days . injection of ghrelin increased food intake , in which significant ( p < .05 ) difference was obtained at the 2nd , 4th and 5th days ( figure 5(a ) ) . the cumulative food intake during the treatment period of 7 days was significantly ( p < .05 ) increased by ghrelin ( figure 5(b ) ) .   at 2 weeks after total gastrectomy , rats were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for 7 continuous days . the 24 hours food intake averaged for the treatment period of 7 days was not significantly different between ghrelin - and saline - treated groups ( figure 5(c ) ) . essentially the same results were obtained in rats at 6 weeks after total gastrectomy ; 24 hours food intake averaged for the treatment period of 7 days was not different between ghrelin - and saline - treated groups ( figure 5(d ) ) . at the 12th week after total gastrectomy one group received daily subcutaneous injection of saline for a week followed by that of ghrelin for the next week . vice versa , the other group received ghrelin for the first week followed by saline for the next week . the amount of 24 hours food intake in rats injected with ghrelin was significantly ( p < .05 ) greater than that with saline ( figure 5(e ) ) . these results indicate that ghrelin injection increases food intake in the later stage of 12 weeks after gastrectomy but not earlier . ghrelin is secreted primarily from the stomach [ 9 , 30 ] , and stimulates food intake and body weight gain . gastrectomy commonly decreases food intake , body weight , fat mass and bone mass , and is also accompanied by a marked reduction in circulating ghrelin levels . however , whether ghrelin levels are causally related to food intake and/or body weight after gastrectomy is not well understood . in the present study , we produced total gastrectomized rat models and examined postoperative changes in ghrelin levels , food intake and body weight . plasma ghrelin levels were markedly reduced by gastrectomy irrespective to whether the reconstruction was performed by b - ii or r - y method . however , the recovery from gastrectomy - induced reductions in food intake and body weight was much greater in r - y than in b - ii group . the results indicate that the recovery from reduced food intake and body weight after gastrectomy is not correlated with circulating ghrelin levels but strongly depends on the reconstruction method . the mechanism for the better recovery of food intake and body weight with r - y method after total gastrectomy remains unclear . ghrelin contents in the duodenum , jejunum and pancreas at 12th postoperative week were not different between b - ii and r - y methods . it is speculated that in b - ii method bile juice may directly reflux the esophagojejunostomy and consequently decrease food intake . alternatively , shorter distance between esophagojejunostomy and jejunojejunostomy in r - y method than in b - ii method may yield smaller reflux of bile juice . it is suggested that selecting the reconstruction method with less reflux of bile juice may contribute to better recovery from the gastrectomy - induced anorexia .   at the 12th week after gastrectomy , ghrelin production was markedly enhanced in the duodenum , the organ known to produce the second largest amount of ghrelin . this result was essentially the same between b - ii and r - y groups , suggesting that the enhanced ghrelin production is not related to the reconstruction method but likely due to the removal of gastric ghrelin . it is therefore suggested that the enhanced ghrelin production in the duodenum could partly compensate for gastrectomy - associated reductions of the circulating ghrelin and its endocrine functions . in another line of experiments , at 12th postoperative week fasting markedly increased plasma ghrelin levels in gastrectomized rats , indicating that the tissue other than stomach , most likely the duodenum , secretes ghrelin in response to fasting , the physiological regulator of ghrelin secretion [ 9 , 32 ] . furthermore , the feeding response to peripheral ghrelin administration was once eliminated by gastrectomy but restored at 12th postoperative week . these data suggest that ghrelin can be released and stimulate food intake under fasted conditions at this later period after gastrectomy . collectively , the markedly upregulated production of ghrelin in the duodenum could contribute to the recovery of food intake and body weight in the later postoperative period .   in this study , since the ghrelin content in the pancreas and its increment due to gastrectomy are much smaller than those in the duodenum , they may neither significantly contribute to the circulating ghrelin levels nor operate endocrine functions . however , previous studies using ghrelin receptor antagonists and ghrelin - deficient mice have demonstrated that ghrelin in the pancreatic islets inhibits insulin release in an autocrine / paracrine manner and consequently regulate blood glucose levels [ 33 , 34 ] . the late postprandial dumping syndrome accompanying gastrectomy is characterized by hypoglycemia principally due to excessive insulin release . the mechanism for excessive insulin release remains unclear , lack of the stomach - derived ghrelin could be implicated . if so , it is possible that upregulated pancreatic ghrelin compensates lack of stomach - derived ghrelin and attenuate insulin release , thereby counteracting the hypoglycemia in the late dumping syndrome . further study is necessary to elucidate the role and mechanism for the upregulation of ghrelin production in the pancreas after gastrectomy . it has been well documented that administration of pharmacological doses of ghrelin to intact animals increases food intake , induces weight gain , and causes obesity [ 9 , 1318 ] . ghrelin injection failed to stimulate food intake in the early stages of 2nd to 6th week after operation , but increased it in the late stage of 12th week . the results suggest that some machinery that links ghrelin reception to feeding dysfunctions after gastrectomy but can be restored . it has been reported that the peripheral injection of ghrelin does not increase food intake after cutting vagus nerve [ 15 , 30 , 35 ] . it is thought that peripheral ghrelin signal is transmitted to the feeding center at least partly via afferent vagal nerves [ 15 , 30 , 35 ] . in our study , it is speculated that regeneration of vagus nerves takes place not immediately but time - dependently , allowing reception of injected ghrelin and transmission of its signal to the feeding center . in addition , chronic hypoghrelinemia in gastrectomized status was reported to induce hypersensitivity to ghrelin as judged by secretion of growth hormone . if this is also the case for the orexigenic action of ghrelin , the hypersensitivity to ghrelin could make the ghrelin injection more potent and/or efficacious in correcting anorexia after gastrectomy . collectively , in the certain period when vagus nerves are regenerated and sensitivity to ghrelin is elevated , ghrelin treatment is expected to effectively stimulate feeding . in fact , the fractional increase of food intake in response to ghrelin injection at 12th postoperative week was similar to that in control rats ( figures 5(e ) versus 5(a ) ) . thus , our results raise a possibility that ghrelin replacement therapy given in later stages could be effective in promoting food intake and correcting some symptoms associated with gastrectomy in humans . an effective treatment of severe obesity is bariatric surgery , in which gastric bypass is a representative method . gastric bypass , as well as gastrectomy , markedly reduces appetite and body weight , suggesting that changes in plasma ghrelin levels are involved . however , studies on changes in plasma ghrelin levels after gastric bypass have yielded inconsistent results ; decreased , unchanged , or increased [ 3840 ] . in our study , better restoration in feeding and body weight after gastrectomy was obtained with r - y than b - ii method , while plasma ghrelin levels were similar . these observations by us and others collectively suggest that feeding and body weight in the earlier period after gastric surgery , are not associated with plasma ghrelin levels but determined by other factors , possibly reconstruction methods associated with specific histological conditions . in summary , recovery from gastrectomy - induced reductions in food intake and body weight is not related to plasma ghrelin levels but depends on the reconstruction method in rats . at postoperational 12th week , ghrelin production is increased in the duodenum and pancreas , and circulating ghrelin level is elevated by fasting , suggesting that ghrelin could be released from extra - stomach tissues and play a compensatory role . ghrelin administration stimulates feeding at postoperational 12th week but not earlier , suggesting that ghrelin treatment in later periods could be effective in treating patients with gastrectomy - induced anorexia and associated symptoms .