obesity is recognized as a worldwide problem , especially for developing insulin resistance and increasing the risk of type 2 diabetes1 . the average body mass index ( bmi ) of japanese diabetic patients has been increasing in recent years , in parallel with an increase in dietary fat intake . gastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) and glucagonlike peptide1 ( glp1 ) are incretins , peptide hormones released from the gastrointestinal tract into circulation in response to nutrient ingestion that potentiate glucosestimulated insulin secretion2 , 3 , 4 . gip is secreted from k  cells located in the upper small intestine ; glp1 is secreted from l  cells located in the lower small intestine and colon . total plasma gip levels in wildtype ( wt ) mice after oral lard administration are much higher than that after oral glucose administration5 . the peak value of plasma gip in response to a highfat meal ( 450  kcal containing 33.3% fat ) is threefold higher than that by 75  g oral glucose tolerance test in human subjects , suggesting that the fat content in a mixed meal strongly stimulates gip secretion6 . gip is considered to increase the volume of adipose tissue by two major pathways : directly by binding to the gip receptor located on the adipocytes7 , 8 and indirectly , by accelerating fat deposition and expansion of fat depots by increasing insulin secretion from pancreatic cells9 . studies of gip receptor knockout mice show gip to be an obesitypromoting factor in highfat diet ( hfd ) conditions10 , and show deletion of gip receptor signaling to cause resistance to dietinduced obesity11 . additionally , we reported that partial reduction of gip alleviates obesity and lessens the degree of insulin resistance without exacerbating glucose tolerance under hfd conditions12 . increased plasma gip levels in obesity have been shown in several studies13 , 14 , 15 , 16 . we previously reported that the plasma gip level after glucose loading is positively correlated with body mass index in healthy subjects17 . furthermore , a report showing that healthy human subjects given high fat food for 2  weeks show increased plasma gip levels without developing obesity , suggesting that gip hypersecretion precedes obesity18 . these findings suggest that there are both acute mechanisms of gip secretion in response to a single administration of fat and chronic mechanisms for hyperproduction of gip under hfd feeding . however , the precise mechanisms of gip secretion and gip production have remained unclear , mainly because of inability to isolate gipproducing k  cells from intestinal epithelium . recently , we generated gipgfp knockin mice , in which k cells are labeled by enhanced green fluorescent protein , and have succeeded in isolating k  cells using a flow cytometry technique . on the basis of microarray analysis of k  cells isolated from gipgfp knockin heterozygous ( gip ) mice , we showed that some factors highly expressed in k  cells are involved in fatinduced gip secretion and gip hypersecretion in dietinduced obesity . fatty acidbinding protein  5 ( fabp5 ) is a 15  kda cytosolic protein with a high affinity to long chain fatty acids , which has been known as an intracellular chaperon transporting long chain fatty acids into various organelles . using microarray analysis , we showed that fabp5 is expressed in murine k  cells , and investigated the physiological function of fabp5 in k  cells5 . immunostaining of intestinal mucosa showed that gippositive cells were totally merged with fabp5positive cells , and that 90% of fabp5positive cells were merged with gippositive cells . to evaluate the acute gip secretory response in fabp5 knockout ( fabp5 ) mice , lard ( 10  ml / kg ) and glucose ( 2  mg / kg ) were injected orally , and plasma glucose and serum levels of gip , glp1 and insulin were measured . plasma glucose , insulin and glp1 levels after both glucose and lard administration were similar in fabp5 mice and fabp5 mice ( figure  1a  c , e  g ) . plasma gip levels after lard injection were significantly lower in fabp5 mice compared with those in fabp5 mice ( figure  1h ) , but there were no significant differences in the results of oral glucose tolerance test ( figure  1d ) . given that fabps are known to function as an intracellular lipid chaperone , we speculated that there would be a mechanism by which fattyacid permeates the cell membrane of k  cells for fabp5associated gip secretion in response to fat ingestion . to examine the significance of micelleaided incorporation of long chain fatty acids into k  cells in the presence of bile , as is the case with absorptive epithelial cells in the small intestine , we evaluated gip secretion after glucose or lard administration in mice subjected to common bile duct ligation ( bdl ) , in comparison with mice subjected to sham operation . although gip secretion after glucose injection remained unaffected by bdl , the secretion after lard injection was seriously diminished in bdl mice . dissociation of the effect of bdl on gip secretion after glucose and lard administration suggests that bile is the crucial factor in gip secretion in response to fat , but not to glucose . as an ex  vivo experiment , the upper half portion of the small intestine was harvested from fabp5 and fabp5 mice , shredded into small pieces , and incubated in conditioned media as follows : 5.5  mmol / l glucose dulbecco 's modified eagle medium as a control , 100  mol / l oleic acid , 4 v / v% of bile and 100  mol / l oleic acid plus 4 v / v% of bile . we found no gip release after 15  min incubation with bile , and a very small increase in gip concentration in the media after the incubation with oleic acid . by contrast , there were marked increases of gip secretion from the samples incubated with oleic acid plus bile , and the fabp5 samples showed a 3.4fold decrease compared with that in fabp5 samples . to assess potential effects of fabp5deficiency on bodyweight and composition , mice were fed hfd for 10  weeks , and wholebody computed tomography scans of hfdfed fabp5 , fabp5 , gipgfp knockin homozygous ( gip)fabp5 and gipfabp5 mice were compared . under hfd feeding conditions , fabp5 mice showed significantly decreased bodyweight gain compared with fabp5 mice , but there was no significant difference in bodyweight between gipfabp5 and gipfabp5 mice , in which gip expression is genetically deleted . wholebody computed tomography scan showed that body fat mass was significantly reduced in fabp5 mice compared with that in fabp5 mice , and that body fat mass in gipfabp5 and gipfabp5 mice was comparable . these results show that fabp5 is involved in fatty acidinduced acute gip secretion , and that it contributes to the development of hfdinduced obesity in a gipdependent manner . oral glucose and lard oil tolerance tests to fatty acidbinding protein  5 ( fabp5 ) knockout mice . concentrations of ( a , e ) blood glucose , ( b , f ) serum insulin , ( c , g ) plasma total glucagonlike peptide1 ( glp1 ) and ( d , h ) plasma total gastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) after oral administration of ( a  d ) 2  g / kg glucose and ( e  g ) 10  ml / kg lard . * * p  <  0.01 between wildtype mice ( wt ; n  =  4 ; green circle ) and fabp5 knockout mice ( fabp5 ko ; n  =  5 ; orange circle ) mice . receptors for longchain fatty acids ( g  proteincoupled receptor [ gpr]40 and gpr120)19 , 20 , shortchain fatty acids ( gpr41 and gpr43)21 and oleoylethanolamide ( gpr119)22 are known to be involved in glp1 secretion . however , the role of fatty acidsensing g  proteincoupled receptors ( gpcrs ) , except for gpr40 and gpr119 , in gip secretion from k  cells remains unclear19 , 22 . to evaluate the expression levels of fatty acidsensing gpcrs in k  cells , we carried out reverse transcription polymerase chain reactions using isolated k  cells from gipgfp knockin mice23 . although gpr119 showed a tendency toward abundance in gfppositive k  cells of the lower small intestine , the difference between gfppositive and gfpnegative cells was not significant . gpr40 and gpr43 were highly expressed in k  cells of the lower small intestine . in contrast , gpr120 was highly expressed in k  cells of the upper small intestine and not of the lower small intestine . we have found that not only k  cell number , but also gip content and gip messenger ribonucleic acid ( mrna ) expression in k  cells are greater in the upper small intestine compared with those in the lower small intestine23 , suggesting that k  cells in the upper small intestine contribute more to nutrientinduced gip secretion than k  cells of the lower small intestine . in such a context , it might be assumed that gpr120 , which is highly expressed in k  cells of the upper small intestine , is crucial for fatty acidinduced gip secretion . to ascertain the role of gpr120 in gip secretion , we measured plasma gip levels after oral lard oil administration in wt mice and gpr120 knockout ( gpr120 ) mice23 . glucoseinduced gip secretion was similar between wt and gpr120 mice ( figure  2a , b ) . in contrast , gip secretion induced by lard oil in gpr120 mice was significantly reduced to 25% of that in wt mice ( figure  2c , d ) . furthermore , we examined the effect of grifolic acid methyl ether , which competitively inhibits longchain fatty acidinduced activation of gpr120 signaling24 , on gip secretion . treatment of mice with grifolic acid methyl ether resulted in decreased lard oilinduced gip secretion , but not in glucoseinduced gip secretion . these results suggest that fatty acids derived from lard oil stimulate gip secretion through gpr120 signaling . gastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) secretion in g  proteincoupled receptor  120 ( gpr120 ) knockout mice . concentrations of total plasma gip after oral administration of ( a ) 2  g / kg glucose and ( c ) 10  ml / kg lard . area under the curve ( auc ) of gip concentrations after oral administration of ( b ) glucose and ( d ) lard . gpr120 ko ; wt , * p  <  0.05 , * * p  <  0.01 between wildtype mice ( wt ; n  =  5 ; green circle / green bar ) and gpr120 knockout mice ( gpr120 ko ; n  =  5 ; orange circle / orange bar ) . several studies have reported that gip secretion is increased in obesity13 , 14 , 15 , 16 . however , the mechanisms involved in gip hypersecretion from k  cells in obesity remain unclear , mainly because of difficulties in separating these cells from other intestinal epithelial cells in  vivo . it has been shown that pancreatic and duodenal homeobox  1 ( pdx1 ) , which is known to be an important transcription factor in pancreatic development and pancreatic cell maturation25 , has a critical role in gip production in k  cells26 , 27 . however , the contribution of pdx1 and other transcription factors in obesityassociated gip hypersecretion has not been confirmed . based on microarray analysis data , we showed that mrna of regulatory factor  x6 ( rfx6 ) is highly expressed in k  cells28 . immunohistochemistry of the small intestine from gipgfp knockin mice confirmed that rfx6expressing cells correspond to gfpexpressing cells , showing that rfx6 is highly expressed in k  cells in the murine small intestine . the rfx gene family of transcription factors was first detected in mammals as regulatory factors that bind to the promoter regions of major histocompatibility complex class ii genes29 ; seven types of rfx ( rfx17 ) have so far been identified . rfx14 and 6 have a dimerization domain30 , 31 , and rfx6 forms homodimers or heterodimers with rfx2 or rfx332 , 33 . serial analysis of gene expression ( sage ) frequency data showed high expression of rfx6 mrna in the pancreas , liver and heart , and reverse transcription polymerase chain reaction analysis showed high expression of rfx6 mrna in the human pancreas and intestine34 . rfx6deficient mice were previously generated , and none of the endocrine cells , excluding pancreatic polypeptideexpressing cells , were detected in the islets of these mice34 . these results suggest that rfx6 plays a critical role in generating the endocrine cells in islets , but it has been unknown whether rfx6 is associated with generation of enteroendocrine cells , such as k  cells . to examine the involvement of rfx6 in gip gene expression , we evaluated gip mrna expression and content under the inhibition of rfx6 expression in  vitro . by treatment with rfx6 small interfering rna , mrna expression and cellular content of gip were significantly decreased in mouse enteroendocrine cell line stc1 cells . rfx6 effectively bound to a fragment of the gip promoter ( 52166512  bp upstream of the gip promoter ) . in the luciferase promoter assay , gip promoter activity of the fragments containing 52166512  bp upstream of the gip promoter was high . these results suggest that rfx6 binds to the region 52166512  bp upstream of the gip promoter , which regulates gip promoter activity . furthermore , we confirmed that gip mrna expression levels were significantly increased in rfx6overexpressing stc1 cells , showing that rfx6 expressed by k  cells plays an important role in gip gene expression . to investigate the mechanisms of gip hypersecretion in hfdinduced obesity in  vivo , gipgfp heterozygous mice were fed a controlfat diet ( cfd ) or hfd , and k  cells were isolated for further evaluation . from 1  week after starting these diets onward , the bodyweight of the hfd group remained significantly higher compared with that of the cfd group . after cfd or hfd feeding for 8  weeks , oral glucose tolerance tests were carried out . gip secretion ( area under the curvegip ) of the hfd group was increased by approximately 1.5fold compared with that of the cfd group . these results show that hfd feeding increases gip secretion and induces obesity in gipgfp heterozygous mice , even though they have only one normal gip gene , indicating that these mice represent a useful model for analysis of the mechanisms involved in the augmentation of gip secretion in hfdinduced obesity . to determine whether gip hypersecretion is caused by an increased number of k  cells in hfdfed gipgfp heterozygous mice , the number of k  cells in the upper small intestine of cfdfed mice and hfdfed mice were estimated and compared . we could not detect an increase of k  cell number in the duodenum or upper small intestine of hfdfed gipgfp heterozygous mice by immunohistochemistry and flow cytometry analysis . in addition , in k  cells purified by using flow cytometry , the expression levels of gip mrna were almost 10fold higher in the hfd group than those in the cfd group . these results show that gip hypersecretion under hfdinduced obesity is not due to an increase in k  cell number , but to an increase of gip mrna expression and content in k  cells . furthermore , the expression levels of rfx6 and pdx1 mrna were significantly increased in k  cells of hfdinduced obese mice compared with those of cfdfed lean mice ( figure  3 ) . in previous studies , it has been reported that pdx1 binds 150  bp upstream of the gip promoter and activates the gip promoter in stc1 cells , and that pdx1 expression is essential for producing gip in k  cells26 , 27 . thus , an increase in rfx6 and pdx1 expressions in k  cells of hfdinduced obese mice is consistent with the in  vitro data showing that rfx6 and pdx1 are involved in gip gene expression . increased expression levels of pancreatic and duodenal homeobox  1 ( pdx1 ) and regulatory factor  x6 ( rfx6 ) messenger ribonucleic acid ( mrna ) in k  cells after highfat diet ( hfd ) feeding . ( a ) concentrations of plasma total gastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) after oral administration of 2  g / kg glucose in controlfat diet ( cfd)fed gipgreen fluorescent protein knockin ( gip 
 ) mice ( n  =  5 ; green circle ) and hfdfed gip 
  mice ( n  =  5 ; orange circle ) . * p  <  0.05 , * * p  <  0.01 . ( b ) number of k  cells in the upper small intestine ( n  =  5 ) . ( c ) gip content in the upper small intestine ( n  =  57 ) . ( d ) expression of gip mrna in k  cells ( n  =  810 ) . ( e ) expression of pdx1 mrna in k  cells ( n  =  810 ) . ( f ) expression of rfx6 mrna in k  cells ( n  =  810 ) . gfppositive cells from cfdfed gip 
  mice ( green bar ) , from hfdfed gip 
  mice ( orange bar ) . , we found that transcription factor rfx6 is highly expressed in k  cells , and is involved in the regulation of gip expression . we also showed that expression of rfx6 and pdx1 is upregulated in the k  cells of hfdinduced obese mice , which suggests that induction of rfx6 as well as pdx1 plays a critical role in gip hypersecretion in hfdinduced obesity . gene analysis of k  cells isolated from gipgfp mice enabled us to identify candidate genes that contribute to acute and chronic mechanisms stimulating gip secretion in response to fat ingestion : fabp5 and gpr120 play crucial roles in acute fatinduced gip secretion , and rfx6 is involved in hypersecretion of gip in hfdinduced obese conditions by increasing gip gene expression ( figure  4 ) . although further , detailed analyses are required to clarify the intracellular signaling of gip secretion and synthesis in response to nutrient ingestion , regulation of gip secretion could provide a novel therapeutic approach to prevent obesity , insulin resistance and subsequent type 2 diabetes . the mechanisms of fatinduced gastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) secretion and increased gip synthesis in response to after highfat diet ( hfd ) feeding . fatty acidbinding protein  5 ( fabp5 ) and g  proteincoupled receptor  120 ( gpr120 ) play crucial role in acute fatinduced gip secretion and regulatory factor  x6 ( rfx6 ) is involved in hypersecretion of gip in hfdinduced obese conditions by increasing gip gene expression . gpcr , g  proteincoupled receptor ; pdx1 , pancreatic and duodenal homeobox  1 . . ni served as a medical advisor for takeda , taisho pharmaceutical , glaxosmithkline and mitsubishi tanabe pharma , and lectured for msd , sanofi , novartis pharma , dainippon sumitomo pharma , kyowa kirin and mitsubishi tanabe pharma , and received payment for his services . eli lilly japan , shiratori pharmaceutical , roche diagnostics and the japan diabetes foundation , and also received a scholarship grant from msd , japan tobacco inc . , nippon boehringer ingelheim , takeda , dainippon sumitomo pharma , astellas pharma , daiichisankyo and mitsubishi tanabe pharma .
abstractgastric inhibitory polypeptide / glucosedependent insulinotropic polypeptide ( gip ) is one of the incretins , which are gastrointestinal hormones released in response to nutrient ingestion and potentiate glucosestimulated insulin secretion . 
 single fat ingestion stimulates gip secretion from enteroendocrine k  cells ; chronic highfat diet ( hfd ) loading enhances gip secretion and induces obesity in mice in a gipdependent manner . 
 however , the mechanisms of gip secretion from k  cells in response to fat ingestion and gip hypersecretion in hfdinduced obesity are not well understood . 
 we generated gipgreen fluorescent protein knockin ( gip 
 gfp/+ ) mice , in which k  cells are labeled by enhanced gipgreen fluorescent protein . 
 microarray analysis of isolated k  cells from gip 
 gfp/+ mice showed that both fatty acidbinding protein  5 and g  proteincoupled receptor  120 are highly expressed in k  cells . 
 single oral administration of fat resulted in significant reduction of gip secretion in both fatty acidbinding protein  5 and g  proteincoupled receptor  120deficient mice , showing that fatty acidbinding protein  5 and g  proteincoupled receptor  120 are involved in acute fatinduced gip secretion . 
 furthermore , the transcriptional factor , regulatory factor  x6 ( rfx6 ) , is highly expressed in k  cells . in vitro experiments using the mouse enteroendocrine cell line , stc1 , showed that gip messenger ribonucleic acid levels are upregulated by rfx6 . 
 expression levels of rfx6 messenger ribonucleic acid as well as that of gip messenger ribonucleic acid were augmented in the k  cells of hfdinduced obese mice , in which gip content in the small intestine is increased compared with that in lean mice fed a control diet . 
 these results suggest that rfx6 is involved in hypersecretion of gip in hfdinduced obese conditions by increasing gip gene expression .