animal testing procedures were approved by the animal care and use committee of the national institute on aging . methods are described in detail in the supplementary material , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0807/dc1 . serial sections ( 10-m thickness ) were cut from tissues containing circumvallate , foliate , and fungiform papillae , using a cryostat ( microm ; thermo scientific ) . to obtain systematic samples without bias throughout the papillae , taste buds are 80100 m in length , therefore sampling every tenth section ensures that no two sections will be from the same taste bud . approximately 100120 taste buds per group were analyzed immunohistochemically as described previously ( 11,29 ) . only nuclear profile the total number of cells in the section was determined by counting the number of to - pro-3stained nuclei present in each taste bud . the percentage of immunoreactive taste cells was calculated by : number of immunoreactive taste cells / total number of taste cells in each taste bud . plasma levels of gut and appetite hormones were measured using a mouse multiplex assay kit ( millipore ) according to the manufacturer 's instructions . quantification of pancreatic immunohistochemistry images was performed in matlab ( mathworks ) as described previously ( 30 ) . hypothalamic protein levels of various factors involved in energy regulation were determined by western blot analyses . vip knockout and wild - type ( n = 8) mice were habituated to the laboratory environment for 30 min each day prior to testing . the same animals were used for both the taste behavioral tests and analyses of physiological parameters ( pancreatic function , gut / appetite plasma hormone analyses ) . test stimuli consisted of various concentrations of sucrose ( 25 , 75 , 150 , 300 , and 600 mmol / l ; fisher scientific ) , sodium chloride ( nacl : 100 , 200 , 400 , 600 , 800 , and 1,000 mmol / l ; sigma ) , denatonium benzoate ( 0.001 , 0.01 , 0.1 , 0.3 , 1 , 5 , and 10 mmol / l ; sigma ) , and citric acid ( 0.01 , 0.1 , 1 , 3 , 5 , 10 , 30 , and 100 mmol / l ; fisher scientific ) . brief - access taste testing took place in a davis ms-160 gustometer ( dilog instruments ) as previously described ( 11,3134 ) . brief - access procedures minimize postingestive effects that may confound other assays such as intake tests ( 33 ) . to test salty , sour , and bitter stimuli responses , mice were water deprived prior to testing . for testing of the sweet stimulus , mice were both food and water restricted ( 2.5 ml of water , 1 g of food ) prior to testing . all mice were tested with a single tastant at various concentrations per trial , to prevent the mice from being affected by prior experience . animal testing procedures were approved by the animal care and use committee of the national institute on aging . methods are described in detail in the supplementary material , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0807/dc1 . methods are described in detail in the supplementary material . serial sections ( 10-m thickness ) were cut from tissues containing circumvallate , foliate , and fungiform papillae , using a cryostat ( microm ; thermo scientific ) . to obtain systematic samples without bias throughout the papillae , taste buds are 80100 m in length , therefore sampling every tenth section ensures that no two sections will be from the same taste bud . approximately 100120 taste buds per group were analyzed immunohistochemically as described previously ( 11,29 ) . only nuclear profile the total number of cells in the section was determined by counting the number of to - pro-3stained nuclei present in each taste bud . the percentage of immunoreactive taste cells was calculated by : number of immunoreactive taste cells / total number of taste cells in each taste bud . plasma levels of gut and appetite hormones were measured using a mouse multiplex assay kit ( millipore ) according to the manufacturer 's instructions . quantification of pancreatic immunohistochemistry images was performed in matlab ( mathworks ) as described previously ( 30 ) . hypothalamic protein levels of various factors involved in energy regulation were determined by western blot analyses . vip knockout and wild - type ( n = 8) mice were habituated to the laboratory environment for 30 min each day prior to testing . the same animals were used for both the taste behavioral tests and analyses of physiological parameters ( pancreatic function , gut / appetite plasma hormone analyses ) . test stimuli consisted of various concentrations of sucrose ( 25 , 75 , 150 , 300 , and 600 mmol / l ; fisher scientific ) , sodium chloride ( nacl : 100 , 200 , 400 , 600 , 800 , and 1,000 mmol / l ; sigma ) , denatonium benzoate ( 0.001 , 0.01 , 0.1 , 0.3 , 1 , 5 , and 10 mmol / l ; sigma ) , and citric acid ( 0.01 , 0.1 , 1 , 3 , 5 , 10 , 30 , and 100 mmol / l ; fisher scientific ) . brief - access taste testing took place in a davis ms-160 gustometer ( dilog instruments ) as previously described ( 11,3134 ) . brief - access procedures minimize postingestive effects that may confound other assays such as intake tests ( 33 ) . to test salty , sour , and bitter stimuli responses , mice were water deprived prior to testing . for testing of the sweet stimulus , mice were both food and water restricted ( 2.5 ml of water , 1 g of food ) prior to testing . all mice were tested with a single tastant at various concentrations per trial , to prevent the mice from being affected by prior experience . gross taste bud morphology and taste cell number were assessed in wild - type and vip knockout mice using classical taste cell type  specific immunocytochemical markers ( supplementary tables 1 and 2 ) . compared with wild - type mice , vip knockout mice showed no gross taste bud abnormalities and expressed similar distribution patterns of taste cell  specific markers ( fig . vip immunoreactivity was present in the taste cells of wild - type animals ( fig . the lack of vip , therefore , did not significantly affect initial taste cell development and number . expression of taste cell markers and vip in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . type ii cell marker ( plc2 ) ( a and b ) , type iii cell marker ( ncam ) ( c and d ) , and type iv cell marker ( shh ) ( e and f ) are expressed in taste cells of both wild - type and vip knockout mice . g and h : vip ( red ) is expressed in a subset of taste cells of wild - type mice . i and j : vip is not expressed within the vip knockout mouse taste bud . ( a high - quality digital representation of this figure is available in the online issue . ) both vpac1/2 receptors were primarily expressed in phospholipase c2 ( plc2)-positive taste cells ( type ii cells : fig . 2 ; supplementary figs . 2 and 3 ) . the presence of vip and its cognate receptors in type ii taste cells suggests that vip signaling is local to the taste bud and could likely affect tongue - localized gustation . coexpression of vip receptors ( vpac1/vpac2 ) and plc2 in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . a  c : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice . d  f : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice . i : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice . l : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice . ( a high - quality digital representation of this figure is available in the online issue . ) vip is coexpressed with the taste transduction marker -gustducin and with the t1r2 gpcr ( 20 ) . the majority of vip - immunoreactive taste cells also coexpresses -gustducin , although a subset also expresses t1r2 . -gustducin and t1r2 are markers for type ii cells , and based on this specific expression pattern , we hypothesized that vip could be involved in the transduction of sweet and bitter stimuli . to investigate this , we first determined the expression patterns in the wild - type and vip knockout mice of additional hormones and receptors known to play a functional role in sweet taste coding , that is , the t1r3 sweet stimulant receptor , glp-1 , and leptin receptor ( ob - rb ) ( fig . f ) . both wild - type and vip knockout mice showed similar expression patterns of t1r3 in the circumvallate papillae ( fig . interestingly , we found that glp-1 expression was significantly increased in the taste cells of vip knockout mice compared with wild type ( fig . leptin receptor expression was markedly reduced in vip knockout mice compared with wild type ( fig . peripheral leptin has been shown to suppress sweet taste preference through its action on the leptin receptors in the taste cells ( 2628 ) . elevation in glp-1 expression and the reduction in leptin receptor expression strongly indicates that vip could play a role in modulating sweet taste . taken together , it is apparent that there are significant differences in the numbers of glp-1 and ob - rb  immunopositive cells between wild - type and vip knockout mice ( fig . 3 g , supplementary table 2 ) . to enhance our understanding of these alterations in the taste cells of vip knockout mice , we next determined whether other systemic alterations in appetite / energy - regulatory hormones occur in vip knockout mice . expression of taste - related proteins in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . a and b : sweet taste receptor ( t1r3 , green ) is expressed in both wild - type and vip knockout mice . c and d : glp-1 ( green ) is expressed in wild - type and vip knockout mice . e and f : leptin receptor ( green ) is expressed in wild - type and vip knockout mice . g : the percentage of immunopositive cells quantified in both wild - type and vip knockout mice for the following markers : plc2 , ncam , shh , t1r3 , glp-1 , and leptin receptor . cells were scored as immunoreactive only if a nuclear profile was present within the cell . ( a high - quality digital representation of this figure is available in the online issue . ) as we detected reduced leptin receptor expression in vip knockout mouse taste cells compared with wild type , we measured plasma leptin levels in these mice . circulating leptin can bind to and activate the leptin receptor on taste cells , where it acts to specifically inhibit gustatory responses to sweet substances , without affecting responses to sour , salty , and bitter substances in lean mice ( 2628 ) . in vip knockout mice , both nonfasting and fasting glucose levels in addition , we noted a significant elevation in fasting insulin in vip knockout mice compared with wild type ( fig . we also found that plasma leptin levels were significantly elevated in vip knockout mice compared with wild type ( fig . 4e ) were not different between wild - type and vip knockout mice , whereas vip knockout mice possessed considerably higher plasma gastrointestinal polypeptide ( fig . 4h ) . plasma pancreatic polypeptide ( pp ) levels between wild - type and vip knockout mice were not significantly different despite a trend for lower pp levels in the vip knockout mice ( fig . 4i ) . vip knockout ( ko ) mice possess alterations in multiple hormones controlling appetite and energy balance . multiplex hormone analysis from plasma samples of wild - type ( wt ) and vip knockout mice reveal multiple distinctions between the two animal groups . a and b : significantly higher nonfasting and fasting glucose occurs in the vip knockout mice . the vip knockout mice also possessed significantly higher plasma insulin ( c ) and leptin ( d ) levels . no significant differences between wild - type and vip knockout for amylin were noted ( e ) . vip knockout mice demonstrated higher levels of plasma gastrointestinal polypeptide ( f ) , glp-1 ( g ) , and peptide tyrosine tyrosine ( pyy ) ( h ) . no significant difference in the plasma levels of pp were noted between wild - type and vip knockout mice ( i ) . each panel depicts means  sem from eight animals in each group . * p < 0.05 , * * p < 0.01 . as vip knockout mice possessed significantly elevated plasma insulin levels compared with wild - type mice ( p < 0.01 ) , we next investigated pancreatic islet size and distribution of -cells and -cells . vip knockout mice showed similar total -cell area compared with wild - type ( fig . no significant difference in the total percentage of - or -cells in the islets ( fig . 5d ) and the number of small- , middle- , and large - sized islets between wild - type and vip knockout mice was observed ( fig . pancreatic islet sizing in vip knockout ( ko ) and wild - type ( wt ) mice . representative pancreatic islets from wild - type ( a ) or vip knockout ( b ) mice demonstrate similar insulin ( red fluorescence ) and glucagon ( green fluorescence ) expression profiles . c : mean islet area ( mm ) measured in wild - type or vip knockout pancreata . d : similar mean -cell (  ) and -cell (  ) percentage in the islets of vip knockout and wild - type mice . e : relative distribution of the different islet sizes ( 1 = small ; 2 = middle ; 3 = large ) for vip knockout and wild - type mice . ( a high - quality digital representation of this figure is available in the online issue . ) as a considerable amount of our molecular / immunocytochemical data suggested a role for vip in taste modulation , we next investigated vip 's role in taste perception . we tested the ability of wild - type and vip knockout mice to detect prototypical taste stimuli , that is , sweet ( sucrose ) , sour ( citric acid ) , salty ( sodium chloride ) , and bitter ( denatonium benzoate ) . vip knockout mice displayed significantly altered concentration - dependent licking for sucrose ( p = 0.0108 between mean half - maximal effective concentration [ ec50 ] values ) and denatonium benzoate ( p = 0.0003 between mean ec50 values ) compared with wild - type mice , demonstrated by their lower ec50 values compared with wild type ( fig . vip knockout mice also showed concentration - dependent changes in licking for citric acid ( p = 0.023 between mean ec50 values ) ( fig . 6b ) , but there was no significant alteration for sodium chloride in the vip knockout mice compared with wild - type mice ( fig . these results indicate that vip plays a specific functional role in modulating taste perception of sweet , bitter , and sour stimuli . altered sweet , bitter , and sour taste responses of wild - type ( wt ) and vip knockout ( ko ) mice in brief - access taste tests ( a  d ) . taste responses , expressed as tastant / water lick ratios and as a function of stimulus concentration , of vip knockout ( closed triangle , dotted line ) and wild - type ( open square , solid line ) to sucrose ( a ) , citric acid ( ca ; b ) , denatonium benzoate ( db ; c ) , and sodium chloride ( nacl ; d ) . our data indicate that there is an alteration in the biochemical , but not structural , nature of the tongue of vip knockout mice , as well as complex alterations in circulating metabolic hormones . one paradoxical finding , however , was the reduction in leptin receptor levels in the tongue . vip knockout mice demonstrate several features reminiscent of insulin resistance ( e.g. , high fasting glucose and insulin levels ) . perhaps this change in taste preference  induced by increased tongue glp-1 levels and reduced leptin receptor levels  could be a compensatory mechanism engendered to ameliorate their dysglycemia by attempting to reduce sugar intake . high circulating levels of leptin would be expected to reduce sweet taste preference ( 25 ) , but with a significant ablation of the lingual ob - rb , this would be prevented . however , if this ob - rb ablation was universal , especially in the hypothalamus , then the animal would likely consume even more food and thus exacerbate its dysglycemic state . we , therefore , assessed the expression level of ob - rb in the hypothalami of wild - type and vip knockout mice . in contrast to the tongue , no difference in ob - rb expression between wild - type and vip knockout hypothalami was observed ( fig . in addition , we found no difference in insulin receptor substrate 2 ( irs2 ) , t1r3 receptor , or glp-1 expression in the hypothalami of wild - type or vip knockout mice ( fig . vip knockout mice showed a small reduction in food intake compared with wild - type mice ( fig . 7f ) and there was no significant difference in body weight between vip knockout and wild - type mice ( fig . thus , it appears that the vip knockout mice possess a complex physiological reaction to the imposed loss of vip , one that may , in part , attempt to ameliorate their dysglycemia . vip knockout mice show an increase in sweet taste preference and have elevated circulating leptin levels , which together could potentially be a mechanism to prevent additional excessive sugary food intake ( summarized in fig . however , this mechanism would succeed only if there was localized lingual loss of the ob - rb , which was observed in the vip - null mice . normal leptin receptor and irs2 expression in the hypothalami of wild - type ( wt ) and vip knockout ( ko ) mice . a : representative western blots of three wild - type ( 13 ) or vip knockout ( 13 ) mice hypothalami . the tissue extracts were probed with specific antisera for the leptin receptor ( ob - rb ) , irs2 , and -actin as a loading control . for the ob - rb blot , a plasma membrane extract was used , whereas cytoplasmic tissue extracts were used for the irs2 and actin blots . b  e : mean  sem of the relative band intensities for the specific western blots measured as relative absorbance units minus background absorbance per square pixel ( au - b / px ) . f : mean food intake ( g ) in wild - type and vip knockout mice . values are expressed as means  sem . a diagrammatic summary of the complex physiological parameters observed in the vip knockout ( ko ) mice compared with the wild - type ( wt ) controls . the vip knockout mice present with a diabetic - like state , and they actively attempt to ameliorate this condition with a series of physiological alterations ( high circulating leptin , low tongue ob - rb expression , high tongue glp-1 expression , normal hypothalamic ob - rb expression ) that  in concert  could attenuate the animals ' euglycemic disruption by enhancing their sweet taste and potentially reducing their desire to consume sweet foods . gross taste bud morphology and taste cell number were assessed in wild - type and vip knockout mice using classical taste cell type  specific immunocytochemical markers ( supplementary tables 1 and 2 ) . compared with wild - type mice , vip knockout mice showed no gross taste bud abnormalities and expressed similar distribution patterns of taste cell  specific markers ( fig . vip immunoreactivity was present in the taste cells of wild - type animals ( fig . the lack of vip , therefore , did not significantly affect initial taste cell development and number . expression of taste cell markers and vip in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . type ii cell marker ( plc2 ) ( a and b ) , type iii cell marker ( ncam ) ( c and d ) , and type iv cell marker ( shh ) ( e and f ) are expressed in taste cells of both wild - type and vip knockout mice . g and h : vip ( red ) is expressed in a subset of taste cells of wild - type mice . i and j : vip is not expressed within the vip knockout mouse taste bud . ( a high - quality digital representation of this figure is available in the online issue . ) both vpac1/2 receptors were primarily expressed in phospholipase c2 ( plc2)-positive taste cells ( type ii cells : fig . 2 ; supplementary figs . 2 and 3 ) . the presence of vip and its cognate receptors in type ii taste cells suggests that vip signaling is local to the taste bud and could likely affect tongue - localized gustation . coexpression of vip receptors ( vpac1/vpac2 ) and plc2 in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . a  c : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice . d  f : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice . i : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice . l : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice . ( a high - quality digital representation of this figure is available in the online issue . ) vip is coexpressed with the taste transduction marker -gustducin and with the t1r2 gpcr ( 20 ) . the majority of vip - immunoreactive taste cells also coexpresses -gustducin , although a subset also expresses t1r2 . -gustducin and t1r2 are markers for type ii cells , and based on this specific expression pattern , we hypothesized that vip could be involved in the transduction of sweet and bitter stimuli . to investigate this , we first determined the expression patterns in the wild - type and vip knockout mice of additional hormones and receptors known to play a functional role in sweet taste coding , that is , the t1r3 sweet stimulant receptor , glp-1 , and leptin receptor ( ob - rb ) ( fig . 3a f ) . both wild - type and vip knockout mice showed similar expression patterns of t1r3 in the circumvallate papillae ( fig . interestingly , we found that glp-1 expression was significantly increased in the taste cells of vip knockout mice compared with wild type ( fig . leptin receptor expression was markedly reduced in vip knockout mice compared with wild type ( fig . peripheral leptin has been shown to suppress sweet taste preference through its action on the leptin receptors in the taste cells ( 2628 ) . elevation in glp-1 expression and the reduction in leptin receptor expression strongly indicates that vip could play a role in modulating sweet taste . taken together , it is apparent that there are significant differences in the numbers of glp-1 and ob - rb  immunopositive cells between wild - type and vip knockout mice ( fig . 3 g , supplementary table 2 ) . to enhance our understanding of these alterations in the taste cells of vip knockout mice , we next determined whether other systemic alterations in appetite / energy - regulatory hormones occur in vip knockout mice . expression of taste - related proteins in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice . a and b : sweet taste receptor ( t1r3 , green ) is expressed in both wild - type and vip knockout mice . c and d : glp-1 ( green ) is expressed in wild - type and vip knockout mice . e and f : leptin receptor ( green ) is expressed in wild - type and vip knockout mice . g : the percentage of immunopositive cells quantified in both wild - type and vip knockout mice for the following markers : plc2 , ncam , shh , t1r3 , glp-1 , and leptin receptor . cells were scored as immunoreactive only if a nuclear profile was present within the cell . values are the means  sem . * * p < 0.01 , * * * p < 0.001 . ( a high - quality digital representation of this figure is available in the online issue . ) as we detected reduced leptin receptor expression in vip knockout mouse taste cells compared with wild type , we measured plasma leptin levels in these mice . circulating leptin can bind to and activate the leptin receptor on taste cells , where it acts to specifically inhibit gustatory responses to sweet substances , without affecting responses to sour , salty , and bitter substances in lean mice ( 2628 ) . in vip knockout mice , both nonfasting and fasting glucose levels in addition , we noted a significant elevation in fasting insulin in vip knockout mice compared with wild type ( fig . we also found that plasma leptin levels were significantly elevated in vip knockout mice compared with wild type ( fig . 4e ) were not different between wild - type and vip knockout mice , whereas vip knockout mice possessed considerably higher plasma gastrointestinal polypeptide ( fig . 4h ) . plasma pancreatic polypeptide ( pp ) levels between wild - type and vip knockout mice were not significantly different despite a trend for lower pp levels in the vip knockout mice ( fig . 4i ) . vip knockout ( ko ) mice possess alterations in multiple hormones controlling appetite and energy balance . multiplex hormone analysis from plasma samples of wild - type ( wt ) and vip knockout mice reveal multiple distinctions between the two animal groups . a and b : significantly higher nonfasting and fasting glucose occurs in the vip knockout mice . the vip knockout mice also possessed significantly higher plasma insulin ( c ) and leptin ( d ) levels . no significant differences between wild - type and vip knockout for amylin were noted ( e ) . vip knockout mice demonstrated higher levels of plasma gastrointestinal polypeptide ( f ) , glp-1 ( g ) , and peptide tyrosine tyrosine ( pyy ) ( h ) . no significant difference in the plasma levels of pp were noted between wild - type and vip knockout mice ( i ) . as vip knockout mice possessed significantly elevated plasma insulin levels compared with wild - type mice ( p < 0.01 ) , we next investigated pancreatic islet size and distribution of -cells and -cells . vip knockout mice showed similar total -cell area compared with wild - type ( fig . no significant difference in the total percentage of - or -cells in the islets ( fig . 5d ) and the number of small- , middle- , and large - sized islets between wild - type and vip knockout mice was observed ( fig . pancreatic islet sizing in vip knockout ( ko ) and wild - type ( wt ) mice . representative pancreatic islets from wild - type ( a ) or vip knockout ( b ) mice demonstrate similar insulin ( red fluorescence ) and glucagon ( green fluorescence ) expression profiles . c : mean islet area ( mm ) measured in wild - type or vip knockout pancreata . d : similar mean -cell (  ) and -cell (  ) percentage in the islets of vip knockout and wild - type mice . e : relative distribution of the different islet sizes ( 1 = small ; 2 = middle ; 3 = large ) for vip knockout and wild - type mice . ( a high - quality digital representation of this figure is available in the online issue . ) as a considerable amount of our molecular / immunocytochemical data suggested a role for vip in taste modulation , we next investigated vip 's role in taste perception . we tested the ability of wild - type and vip knockout mice to detect prototypical taste stimuli , that is , sweet ( sucrose ) , sour ( citric acid ) , salty ( sodium chloride ) , and bitter ( denatonium benzoate ) . vip knockout mice displayed significantly altered concentration - dependent licking for sucrose ( p = 0.0108 between mean half - maximal effective concentration [ ec50 ] values ) and denatonium benzoate ( p = 0.0003 between mean ec50 values ) compared with wild - type mice , demonstrated by their lower ec50 values compared with wild type ( fig . vip knockout mice also showed concentration - dependent changes in licking for citric acid ( p = 0.023 between mean ec50 values ) ( fig . 6b ) , but there was no significant alteration for sodium chloride in the vip knockout mice compared with wild - type mice ( fig . taken together , these results indicate that vip plays a specific functional role in modulating taste perception of sweet , bitter , and sour stimuli . altered sweet , bitter , and sour taste responses of wild - type ( wt ) and vip knockout ( ko ) mice in brief - access taste tests ( a  d ) . taste responses , expressed as tastant / water lick ratios and as a function of stimulus concentration , of vip knockout ( closed triangle , dotted line ) and wild - type ( open square , solid line ) to sucrose ( a ) , citric acid ( ca ; b ) , denatonium benzoate ( db ; c ) , and sodium chloride ( nacl ; d ) . our data indicate that there is an alteration in the biochemical , but not structural , nature of the tongue of vip knockout mice , as well as complex alterations in circulating metabolic hormones . one paradoxical finding , however , was the reduction in leptin receptor levels in the tongue . vip knockout mice demonstrate several features reminiscent of insulin resistance ( e.g. , high fasting glucose and insulin levels ) . perhaps this change in taste preference  induced by increased tongue glp-1 levels and reduced leptin receptor levels  could be a compensatory mechanism engendered to ameliorate their dysglycemia by attempting to reduce sugar intake . high circulating levels of leptin would be expected to reduce sweet taste preference ( 25 ) , but with a significant ablation of the lingual ob - rb , this would be prevented . however , if this ob - rb ablation was universal , especially in the hypothalamus , then the animal would likely consume even more food and thus exacerbate its dysglycemic state . we , therefore , assessed the expression level of ob - rb in the hypothalami of wild - type and vip knockout mice . in contrast to the tongue , no difference in ob - rb expression between wild - type and vip knockout hypothalami was observed ( fig . in addition , we found no difference in insulin receptor substrate 2 ( irs2 ) , t1r3 receptor , or glp-1 expression in the hypothalami of wild - type or vip knockout mice ( fig . vip knockout mice showed a small reduction in food intake compared with wild - type mice ( fig . 7f ) and there was no significant difference in body weight between vip knockout and wild - type mice ( fig . thus , it appears that the vip knockout mice possess a complex physiological reaction to the imposed loss of vip , one that may , in part , attempt to ameliorate their dysglycemia . vip knockout mice show an increase in sweet taste preference and have elevated circulating leptin levels , which together could potentially be a mechanism to prevent additional excessive sugary food intake ( summarized in fig . however , this mechanism would succeed only if there was localized lingual loss of the ob - rb , which was observed in the vip - null mice . normal leptin receptor and irs2 expression in the hypothalami of wild - type ( wt ) and vip knockout ( ko ) mice . a : representative western blots of three wild - type ( 13 ) or vip knockout ( 13 ) mice hypothalami . the tissue extracts were probed with specific antisera for the leptin receptor ( ob - rb ) , irs2 , and -actin as a loading control . for the ob - rb blot , a plasma membrane extract was used , whereas cytoplasmic tissue extracts were used for the irs2 and actin blots . b  e : mean  sem of the relative band intensities for the specific western blots measured as relative absorbance units minus background absorbance per square pixel ( au - b / px ) . f : mean food intake ( g ) in wild - type and vip knockout mice . values are expressed as means  sem . a diagrammatic summary of the complex physiological parameters observed in the vip knockout ( ko ) mice compared with the wild - type ( wt ) controls . the vip knockout mice present with a diabetic - like state , and they actively attempt to ameliorate this condition with a series of physiological alterations ( high circulating leptin , low tongue ob - rb expression , high tongue glp-1 expression , normal hypothalamic ob - rb expression ) that  in concert  could attenuate the animals ' euglycemic disruption by enhancing their sweet taste and potentially reducing their desire to consume sweet foods . we have demonstrated that vip knockout mice possess alterations in their taste cells , circulating metabolic hormones , and taste preferences . vip and its cognate receptors are expressed in the type ii taste cells of the circumvallate papillae . vip knockout mice demonstrated normal gross taste bud morphology ( figs . 1 and 2 ) however , several key biochemical and pharmacologic alterations in their tongues were seen , that is , vip knockout compared with wild - type mice exhibited significant increases and decreases in glp-1 and leptin receptor , respectively , in taste cells ( fig . that glp-1 in taste cells enhances sweet taste perception , this strongly indicated to us that taste cell vip signaling may be involved in sweet taste perception . we demonstrated that vip knockouts possess a greater preference for sweet compounds compared with wild - type mice ( fig . vip knockout mice also exhibited concentration - dependent changes in licking bitter stimuli and sour stimuli , but there was no change in salt perception compared with wild - type mice . it is interesting to note that vpac1/2 receptors were expressed on type ii taste cells . receptor systems responsive to bitter , sweet , and umami stimuli use similar signal transduction systems that themselves are narrowly tuned and achieve functional specificity by expression in nonoverlapping subsets of type ii cells . interestingly , in this study , compared with our previous study ( 11 ) , both vip knockout and wild - type mice presented much higher sucrose / water lick ratios than glp-1 receptor knockout ( glp-1r knockout ) mice and their wild - type controls . it is unsurprising that distinct strains of mice possess different tastant responses , and this observation may be important for future experimental consideration of specific strains to facilitate efficient and sensitive detection of alterations in different taste modalities . one unexpected finding in our vip knockout mice was the significant reduction in leptin receptor ( ob - rb ) expression in the taste cells ( fig . 3f and g ) . circulating leptin can activate leptin receptors on taste cells and specifically inhibit gustatory responses to sweet compounds ( 25 ) . hence , the significant reduction in taste cell leptin receptor expression in vip knockout mice may , in part , account for their heightened preference for sweet tastants . loss of the ob - rb would reduce any inhibitory action that circulating leptin would have upon sweet taste . this finding was interesting in light of our data showing that circulating levels of leptin in vip knockout mice were considerably higher than those in wild type . therefore , despite the presence of high concentrations of this sweet taste suppressor , the reduction of taste cell leptin receptor was able to facilitate enhanced sweet sensation by preventing inhibitory leptin signaling activity in the tongue . the elevated sweet preference of the vip knockout mice may also stem from the elevated levels of taste cell glp-1 . we have previously shown that glp-1 's ability to activate the glp-1r in the tongue strongly modulates sweet taste in mice ( 11 ) . increased levels of glp-1 could , therefore , potentiate glp-1r activity in the tongue , especially considering our previous findings that the enzyme that degrades glp-1 in the peripheral circulation ( dipeptidyl peptidase-4 ) is not present in taste cells of mice ( 11 ) . the precise temporal nature of ob - rb loss , peripheral increase in leptin levels , or elevations in taste cell glp-1 remains to be determined , but our data suggest that there is a strong connection between peripheral energy  regulatory hormones and lingual preference for foodstuffs . from our data , because of the multiple sites and mechanisms of action of vip in mammals , it is unsurprising that the metabolic phenotype of vip knockout mice is complex . hence , despite increased leptin and pyy levels in vip knockout mice there was no dramatic diminution of body weight or food intake . this may , in part , be due to removal of an inhibitory action of vip upon food intake , demonstrated in multiple species including mice ( 35 ) . therefore , the resultant phenotype of the vip knockout mice may be truly an intricate mixture of alterations of metabolic food desire and digestion . in our study , several lines of evidence indicate a strong local taste cell action of vip . first , vip knockout mice demonstrated concentration - dependent changes in licking for sweet , bitter , and sour tastants , but not salt , indicating that there was not a generalized , widespread positive effect on all taste modalities . second , vip knockout and wild - type mice responded similarly to the salty taste stimulus ; therefore , vip knockout mice had no difficulty in learning and completing the taste test . lastly , vip and vip receptors are specifically localized within sweet- and bitter - sensitive taste cells , consistent with the specific taste alterations we observed . further reinforcing a selective local action of vip , we found no vip immunoreactivity in type i cells . it has been demonstrated that amiloride - sensitive na channels are expressed in type i cells of the fungiform papillae , suggesting that these taste cells could play a role in transducing salty taste stimuli ( 36 ) . in line with this finding , we found that there was no difference in salt perception between vip knockout and wild - type mice . thus , it would be reasonable to propose that vip signaling in the taste bud plays a local role in modulating sweet , bitter , and sour taste stimuli . there are numerous potential mechanisms by which vip may affect both sweet and bitter stimuli . sweet and bitter signal transduction is commonly mediated by plc mechanisms involving taste receptor activation , as well as through adenylate cyclase vpac1/2 receptors are coupled to multiple g - protein effectors to activate phospholipase d , plc , and adenylate cyclase . vip stimulation of vpac receptors would therefore likely compete for availability of the plc and adenylate cyclase signaling pathways , potentially diluting the functional signal transduction efficacy of the primary taste receptors . we have previously demonstrated that receptor stimulation of multiple related receptor systems can result in strong inhibitory effects that are profound and long - lasting ( 37 ) . genetic removal of the stimulating ligand will result in a diminution in the number of active vpac1/2 receptors , reducing any diluting competitive actions upon the taste receptor 's signaling capacity . future experimentation upon the signaling efficiency of taste receptors in such an environment may shed light on this hypothesis . the ability of vip ablation to affect not only sweet and bitter taste perception , but also the response to a sour stimulus may be , in part , due to a role of vip in controlling cell - cell signaling between taste cells and the type iii (  presynaptic  ) cells possessing the putative sour receptor ( pkd2l1 ) . recent data have shown that although presynaptic cells do not possess tastant - sensitive gpcrs ( 38 ) , they still respond to tastant stimulation ( 5 ) . communication between taste cells and presynaptic cells is thought to involve atp and serotonin secretion from the taste cells ( 39 ) . in many areas of the gut , vip is coreleased with both atp and nitric oxide ( no ) to exert relaxatory effects on smooth muscle ( 40 ) . no is commonly secreted from postsynaptic nervous tissue to act upon presynaptic neurons to control neurotransmitter release , and no has also been associated with taste transduction ( 41 ) . therefore , it may be possible that in the tongue vip may act in an analogous manner as in other parts of the digestive tract ( i.e. , in a concerted manner with no and atp ) . presynaptic cell communication , resulting in the reduction in sour taste we observed in vip knockout mice . we have demonstrated a complex physiological pattern of changes in vip knockout mice that superficially may seem unconnected but together form a coherent and rational reactive mechanism to maintain energy homeostasis . they also possess significantly higher levels of circulating leptin but a striking reduction in ob - rb expression in the taste cells , while maintaining normal levels of ob - rb and other energy - regulatory factors ( glp-1 , t1r3 ) in the hypothalamus . the loss of vip in our mice induces a dysglycemic state , that is , high resting glucose paired with a high insulin level . with knockout animals that survive birth , it is likely that they have physiologically adapted to counteract their deficits . for example , the lack of alteration of islet -cell mass in the vip knockout mice may be compensated for by the nonselective activity of structurally similar peptides , for example , pituitary adenylate cyclase activating polypeptide , which can activate vpac2 receptors in the vip knockout pancreata , substituting for vip activity ( 18 ) . it would be interesting to speculate that reactive actions in vip knockout mice have attempted to ameliorate their diabetic - like pathology by increasing their sweet preference ( through reduced inhibitory leptin receptor activity and increased glp-1 expression in the tongue ) so as not to overconsume carbohydrates , while also trying to dampen their overall appetite ( increased peripheral circulating leptin with normal hypothalamic ob - rb expression ) ( fig . central nervous system vip is antagonistic to dopamine and serotonin nervous activity ( 42,43 ) . both of these transmitters control food reward responses ( 44,45 ) , and therefore vip knockout mice may possess an elevated reward response , curtailing their intake and preventing weight increases . human analogy of vip knockout mice may be seen in  supertasters  ( possessing increased sweet and bitter sensitivity ) who tend to be more lean and perhaps eat less than nonsupertasters ( 46 ) . however , it is unclear whether the vip knockout mice would not have gained weight if their chow was sweetened with sucrose , and more studies are needed to explore this further . thus , an interesting hypothesis would be that these multiple concerted responses in the vip knockout mice suggest that the tongue could potentially act as a vessel for the animal to modulate its energy intake to correct any endogenous glycemic imbalances . in this way , we could potentially view the tongue as a sensory mechanism that is bidirectionally regulated , and thus forms a bridge between available foodstuffs and the intricate metabolic hormonal balance in the animal itself . , it is becoming apparent that there is a strong link between gustation and peripheral energy balance . hormonal signaling within the taste buds of the tongue is likely to influence food intake and peripheral glucose homeostasis ( 15 ) . for example , hormones such as glp-1 and leptin , crucial to the control of peripheral somatic energy balance / appetite , are now thought to exert a strong local effect on the tongue with respect to food perception . carbohydrates are among the most vital food stuffs for mammalian survival , and it would make evolutionary sense for mammals to have evolved multimodal carbohydrate sensing mechanisms . we feel that gaining a greater appreciation for which endocrine factors are present within taste cells and their putative roles in taste perception will shed much - needed light on how taste perception is linked to peripheral somatic energy balance . this knowledge will be invaluable for finding novel therapeutic targets and agents for the treatment of disorders such as metabolic syndrome and type 2 diabetes .