human glp-1 fragment 736 amide , exendin-4 , nicotinamide , and resveratrol were obtained from sigma ( st . louis , mo ) . rpmi-1640 medium , fcs , and other culture media were purchased from invitrogen ( burlington , on , canada ) . antiacetyl - lysine and anti - sirt1 antibodies were obtained from millipore ( bedford , ma ) . ins832/13 cells ( 24 ) were grown in rpmi-1640 medium supplemented with 10 mmol / l hepes , 10% heat - inactivated fcs , 2 mmol / l l - glutamine , 1 mmol / l sodium pyruvate , 50 mol / l -mercaptoethanol , 100 iu / ml penicillin , and 100 g / ml streptomycin at 37c in a humidified 5% co2 atmosphere . cells at 70% confluence were washed with phosphate - buffered saline and preincubated in serum - free rpmi-1640 medium supplemented with 3 mmol / l glucose and 0.1% bsa ( sigma ) for at least 4 h before treatment . rat islets were isolated from male wistar rats ( 250 g ) by collagenase digestion . human islets were isolated from organ donors at the department of surgery , montreal general hospital , mcgill university health center , montreal , quebec , canada ( three separate donors were received ) . donors were between ages 42 and 65 , and none had a history of diabetes or metabolic disorder . islets were isolated by digestion with liberase ci ( boehringer mannheim , indianapolis , in ) followed by purification over a ficoll gradient using a core 2991 cell processor ( cobe bct , denver , co ) . proteins were extracted and quantified by bca assay ( roche , indianapolis , in ) . foxo1 protein complexes were immunoprecipitated from 1 mg of total protein extracts and subjected to electrophoresis on 8 or 10% polyacrylamide gels . cells were fixed to isolate intact chromatin and sheered in 200-base pair fragments , and sirt1 dna / protein complexes were immunoprecipitated using an anti - sirt1 antibody ( millipore ) . bound dna was amplified by pcr using sequence - specific primers designed for amplification of the forkhead binding site region of the foxa2 promoter ( 16 ) . cells were treated for 15 min , and sirt1 activation was measured using a commercially available fluorometric assay ( biomol , plymouth meeting , pa ) . in brief , the kit uses a sirt1 substrate consisting of amino acids 379382 of human p53 ( arg - his - lys - lys[ac ] ) . the assay 's fluorescence signal is generated in proportion to the amount of deacetylation of lys-382 , a known in vivo target of sirt1 . cell extracts were incubated for 10 min at 37c with a 25 mol / l concentration of the fluorogenic sirt1 substrate . reactions were stopped by the addition of 1 mmol / l nicotinamide , and the fluorescence resonance energy transfer signal was determined . nad , nadh , and atp levels were measured in cell extracts using commercial kits ( nad / nadh quantification kit and atp fluorometric kit from biovision , mountain view , ca ) . nicotinamide phosphoribosyltransferase ( nampt ) activity was measured by the conversion of c - labeled nicotinamide to [ c]nicotinamide mononucleotide as described previously ( 25 ) . in brief , cell lysates ( 10 g total protein ) were incubated with atp , 5-phosphoribosyl-1-pyrophosphate , and [ c]nicotinamide for 30 min ; filtered through glass fiber filters ; and washed with acetone . cells were transfected with either control green fluorescent protein ( gfp ) , wild - type sirt1 , or a catalytically inactive sirt1 mutant ( 26 ) using lipofectamine 2000 ( invitrogen ) as described previously ( 16 ) . the following day , proliferation of transfected cells was evaluated using brdu incorporation assay ( roche ) . in brief , cells were treated for 24 h in the absence or presence of 1 nmol / l glp-1 , and brdu was added to the culture medium for the last 60 min of the incubation period . wild - type and sirbaco transgenic mice ( 27 ) aged 612 months were treated with daily intraperitoneal injections of exendin-4 ( 10 nmol / kg ) or saline for 7 days ( n = 5 each ) . mice were killed , and pancreas sections were processed for insulin and ki67 immunohistochemistry to assess -cell proliferation . cross - sectional islet area was measured using image pro plus software ( media cybernetics , silver spring , md ) . results are presented as relative -cell areas ( i.e. , percent of insulin + areas relative to total pancreas areas ) . for each animal , at least four sections spaced 80 m apart were studied . human glp-1 fragment 736 amide , exendin-4 , nicotinamide , and resveratrol were obtained from sigma ( st . louis , mo ) . rpmi-1640 medium , fcs , and other culture media were purchased from invitrogen ( burlington , on , canada ) . antiacetyl - lysine and anti - sirt1 antibodies were obtained from millipore ( bedford , ma ) . ins832/13 cells ( 24 ) were grown in rpmi-1640 medium supplemented with 10 mmol / l hepes , 10% heat - inactivated fcs , 2 mmol / l l - glutamine , 1 mmol / l sodium pyruvate , 50 mol / l -mercaptoethanol , 100 iu / ml penicillin , and 100 g / ml streptomycin at 37c in a humidified 5% co2 atmosphere . cells at 70% confluence were washed with phosphate - buffered saline and preincubated in serum - free rpmi-1640 medium supplemented with 3 mmol / l glucose and 0.1% bsa ( sigma ) for at least 4 h before treatment . rat islets were isolated from male wistar rats ( 250 g ) by collagenase digestion . human islets were isolated from organ donors at the department of surgery , montreal general hospital , mcgill university health center , montreal , quebec , canada ( three separate donors were received ) . donors were between ages 42 and 65 , and none had a history of diabetes or metabolic disorder . islets were isolated by digestion with liberase ci ( boehringer mannheim , indianapolis , in ) followed by purification over a ficoll gradient using a core 2991 cell processor ( cobe bct , denver , co ) . proteins were extracted and quantified by bca assay ( roche , indianapolis , in ) . foxo1 protein complexes were immunoprecipitated from 1 mg of total protein extracts and subjected to electrophoresis on 8 or 10% polyacrylamide gels . cells were fixed to isolate intact chromatin and sheered in 200-base pair fragments , and sirt1 dna / protein complexes were immunoprecipitated using an anti - sirt1 antibody ( millipore ) . bound dna was amplified by pcr using sequence - specific primers designed for amplification of the forkhead binding site region of the foxa2 promoter ( 16 ) . cells were treated for 15 min , and sirt1 activation was measured using a commercially available fluorometric assay ( biomol , plymouth meeting , pa ) . in brief , the kit uses a sirt1 substrate consisting of amino acids 379382 of human p53 ( arg - his - lys - lys[ac ] ) . the assay 's fluorescence signal is generated in proportion to the amount of deacetylation of lys-382 , a known in vivo target of sirt1 . cell extracts were incubated for 10 min at 37c with a 25 mol / l concentration of the fluorogenic sirt1 substrate . reactions were stopped by the addition of 1 mmol / l nicotinamide , and the fluorescence resonance energy transfer signal was determined . nad , nadh , and atp levels were measured in cell extracts using commercial kits ( nad / nadh quantification kit and atp fluorometric kit from biovision , mountain view , ca ) . nicotinamide phosphoribosyltransferase ( nampt ) activity was measured by the conversion of c - labeled nicotinamide to [ c]nicotinamide mononucleotide as described previously ( 25 ) . in brief , cell lysates ( 10 g total protein ) were incubated with atp , 5-phosphoribosyl-1-pyrophosphate , and [ c]nicotinamide for 30 min ; filtered through glass fiber filters ; and washed with acetone . cells were transfected with either control green fluorescent protein ( gfp ) , wild - type sirt1 , or a catalytically inactive sirt1 mutant ( 26 ) using lipofectamine 2000 ( invitrogen ) as described previously ( 16 ) . the following day , proliferation of transfected cells was evaluated using brdu incorporation assay ( roche ) . in brief , cells were treated for 24 h in the absence or presence of 1 nmol / l glp-1 , and brdu was added to the culture medium for the last 60 min of the incubation period . wild - type and sirbaco transgenic mice ( 27 ) aged 612 months were treated with daily intraperitoneal injections of exendin-4 ( 10 nmol / kg ) or saline for 7 days ( n = 5 each ) . mice were killed , and pancreas sections were processed for insulin and ki67 immunohistochemistry to assess -cell proliferation . cross - sectional islet area was measured using image pro plus software ( media cybernetics , silver spring , md ) . results are presented as relative -cell areas ( i.e. , percent of insulin + areas relative to total pancreas areas ) . for each animal , statistical analyses were performed with spss ( spss inc . , chicago , il ) using anova . we have previously shown that inhibition of the forkhead transcription factor foxo1 mediates the pleiotropic action of the glucoincretin hormone glp-1 ( 10 ) . foxo1 is subject to various posttranslational modifications , including akt - dependent phosphorylation and sirt1-mediated deacetylation . here , we sought to test the hypothesis that glp-1 inhibits sirt1 to enhance foxo1 acetylation and to stimulate -cell mass expansion . we first evaluated foxo1 acetylation and binding to sirt1 by western blot in ins832/13 cells . we resorted to the use of a pancreatic -cell line because of the high amount of biological material required to perform the assays . ins cells were incubated in serum - free medium , a condition known to activate sirt1 , in the presence or absence of glp-1 and the sirt1 activators h2o2 or resveratrol . figure 1a and b shows that glp-1 induced foxo1 acetylation , an action prevented by resveratrol but not by h2o2 . coimmunoprecipitation experiments revealed that glp-1 inhibited the physical association between foxo1 and sirt1 ( fig . the action of glp-1 on foxo1 acetylation was transient , with a maximal effect observed between 15 and 45 min . 1c ) . taken together , these results suggest that glp-1 inhibits sirt1-mediated foxo1 deacetylation . a : foxo1 acetylation and binding to sirt1 were evaluated by western blot after immunoprecipitation of foxo1 protein complexes . ins832/13 cells were incubated in serum - free medium in the presence or absence of glp-1 ( 10 nmol / l ) , with or without the sirt1 activators resveratrol ( 10 mol / l ) and h2o2 ( 25 mol / l ) for 45 min . b : densitometry analysis of five different experiments as shown in a. * p < 0.05 . c : foxo1 acetylation was evaluated as described in a after the indicated periods of time . sirt1 was immunoprecipitated from cross - linked chromatin extracted from ins832/13 cells treated with or without glp-1 for 45 min . eluted dna was pcr - amplified using oligonucleotides flanking the forkhead binding site in the rat foxa2 promoter . e : western blots showing the subcellular localization of sirt1 and foxo1 after 45-min incubation with either 10 nmol / l glp-1 ( glp ) or 10 mol / l resveratrol . chromatin immunoprecipitation assay was performed to assess whether sirt1 is recruited to the promoter region of foxo1 target genes . figure 1d shows that sirt1 was physically associated to a region of the foxa2 promoter encompassing the forkhead binding site ( 10 ) . this result indicates that sirt1 binding to the foxa2 promoter mirrors that of foxo1 , as we have previously demonstrated ( 10 ) . altogether , these findings indicate that sirt1 interacts with foxo1 and is recruited to the promoter of foxo1 target genes in a glp-1inhibitable manner . these data suggest a role for sirt1 in the transcriptional response to the glucoincretin hormone in -cells . we next performed subcellular fractionation to address whether glp-1 provokes sirt1 nuclear exclusion , as it does for foxo1 ( fig . our results show that sirt1 is predominantly nuclear in ins cells and that its subcellular localization was not altered by glp-1 treatment . conversely , glp-1 induced cytoplasmic translocation of foxo1 , as we have published previously ( 10 ) . we next sought to directly test whether glp-1 affects sirt1 activity using an in vitro deacetylase assay . figure 2a shows that glp-1 blunted sirt1 activity to the same extent as the sirt1 inhibitor nicotinamide . conversely , sirt1 activity was increased in resveratrol - treated cells . because sirt1 activity is dependent on nad , we measured changes in the nad - to - nadh ratio in response to glp-1 treatment in ins cells and isolated human islets . figure 2b shows that glp-1 decreased the nad - to - nadh ratio by 30% in ins cells , an action mimicked by the addition of 10% serum . conversely , resveratrol increased the nad - to - nadh ratio by 60% . both glp-1 and serum also elicited a reduction in the nad - to - nadh ratio in human islets ( fig . 2c ) , whereas the effect of resveratrol ( 25% ) did not reach statistical significance . the reduction in nad availability provides a possible mechanism by which glp-1 could acutely regulate sirt1 activity . to directly test this possibility , we supplemented the assay buffer with 450 mol / l nad ( fig . supplementation of nad suppressed glp-1 as well as serum inhibition of sirt1 activity . in an effort to unravel the mechanism by which glp-1 could alter the nad - to - nadh ratio , we investigated its action on the activity of nampt , the rate - limiting enzyme for the biosynthesis of nad , and on the cellular atp content . 2e ) , thereby revealing a novel mechanism by which glp-1 could decrease the nad - to - nadh ratio . fk-866 ( 10 nmol / l ) , a specific nampt pharmacological inhibitor , was used as a positive control . 2f ) , consistent with a recent publication by peyot et al . ( 28 ) . it is interesting that the addition of serum did not alter nampt activity but significantly increased the cellular atp concentration . a : cells were incubated in the presence or absence of 10 nmol / l glp-1 , 10 mmol / l nicotinamide ( nam ) , or 10 mol / l resveratrol ( res ) . proteins were extracted after 15 min to perform the in vitro sirt1 deacetylase assay using a fluorogenic sirt1 substrate . b and c : nad and nadh levels were determined in extracts from cells ( b ) or human islets ( c ) incubated in the presence or absence of 10 nmol / l glp-1 , 10% serum , or 10 mol / l resveratrol for 15 min . d : sirt1 activity was measured as described in a with the modification that the assay buffer was supplemented with 25 mol / l nad . e : nampt activity was measured by the conversion of c - labeled nicotinamide to [ c]nicotinamide mononucleotide . the specific nampt pharmacological inhibitor fk-866 ( 10 we next measured sirt1 expression by quantitative pcr after overnight incubation in serum - free medium in the absence or presence of glp-1 ( fig . our data show that glucose and serum deprivation , a condition mimicking calorie restriction , increased sirt1 expression by fivefold compared with complete growth medium in ins cells . 3b ) . both glp-1 and serum decreased sirt1 expression in rat islets as well , although the detected changes were more modest . thus , sirt1 expression was repressed by 30% in the presence of glp-1 and by 40% in response to serum . we assessed sirt1 protein levels by western blot to test whether alterations in sirt1 expression were translated into changes in protein levels . figure 3c and d shows that glp-1 decreased sirt1 protein levels by 70% in ins cells , an effect mimicked by serum . a and b : cells ( a ) and isolated rat islets ( b ) were incubated in serum - free medium in the absence or presence of glp-1 or left untreated in complete growth medium for 24 h. sirt1 expression levels were measured by quantitative pcr and normalized to actin . c : sirt1 protein levels were evaluated by western blot in ins cells treated as described in a and b. d : densitometry analysis of three different experiments as shown in a and b. * p < 0.05 . to test whether sirt1 inactivation mediates glp-1 action on -cell proliferation , we sought to perform sirt1 gain of function both in vitro and in vivo . ins832/13 cells were either transfected with wild - type sirt1 , a catalytically inactive sirt1 mutant , or with control gfp , and -cell replication was evaluated by brdu incorporation . transfection of the cells with sirt1 led to a fivefold increase in protein levels as compared with gfp ( fig . overexpression of wild - type sirt1 inhibited glp-1induced -cell proliferation but failed to significantly affect the effect of serum ( fig . overexpression of a catalytically inactive sirt1 mutant did not significantly alter -cell proliferation , indicating that the effects of sirt1 on -cell proliferation are dependent on its deacetylase activity . a : sirt1 protein levels were evaluated by western blot after transfection with wild - type ( wt ) or dominant - negative ( dn)-sirt1 . b : the effect of sirt1 overexpression on -cell proliferation was evaluated by brdu incorporation . ins832/13 cells were transfected with wt - sirt1 , dn - sirt1 , or control gfp for 24 h and then incubated in the presence or absence of glp-1 ( 10 nmol / l ) or 10% serum for an additional 24-h period . results represent mean  sem of three separate experiments carried out in triplicate . * p < 0.05 compared with the respective control . to demonstrate the implication of sirt1 in glp-1 action in vivo , we determined whether the effects of glp-1 on -cell mass were curtailed in transgenic mice with increased dosage of sirt1 ( sirbaco mice ) as previously described ( 27 ) . thus , mice were administered with either saline or the long - acting glp-1 analog exendin-4 ( 10 nmol / kg ) daily for 7 days . cross - sectional -cell areas were estimated by morphometry of pancreatic sections stained for insulin ( fig . exendin-4 administration increased the relative -cell area by 2.5-fold and stimulated -cell replication by 50% in wild - type animals . however , glp-1 effects on both -cell area and -cell replication were abolished in sirbaco mice . taken together , our results indicate that sirt1 gain of function prevents the mitogenic action of glp-1 and identify sirt1 as a negative regulator of -cell mass expansion . wild - type ( wt ) or sirbaco transgenic mice received daily injections of exendin-4 ( 10 nmol / kg body wt ) or saline for 7 days . pancreatic -cell mass and -cell replication were evaluated by morphometry and immunohistochemistry with anti - insulin and anti - ki67 antisera . a and b : the relative cross - sectional islet area ( a ) and the percentage of ki67-positive -cells ( b ) are shown . we have previously shown that inhibition of the forkhead transcription factor foxo1 mediates the pleiotropic action of the glucoincretin hormone glp-1 ( 10 ) . foxo1 is subject to various posttranslational modifications , including akt - dependent phosphorylation and sirt1-mediated deacetylation . here , we sought to test the hypothesis that glp-1 inhibits sirt1 to enhance foxo1 acetylation and to stimulate -cell mass expansion . we first evaluated foxo1 acetylation and binding to sirt1 by western blot in ins832/13 cells . we resorted to the use of a pancreatic -cell line because of the high amount of biological material required to perform the assays . ins cells were incubated in serum - free medium , a condition known to activate sirt1 , in the presence or absence of glp-1 and the sirt1 activators h2o2 or resveratrol . figure 1a and b shows that glp-1 induced foxo1 acetylation , an action prevented by resveratrol but not by h2o2 . coimmunoprecipitation experiments revealed that glp-1 inhibited the physical association between foxo1 and sirt1 ( fig . the action of glp-1 on foxo1 acetylation was transient , with a maximal effect observed between 15 and 45 min . 1c ) . taken together , these results suggest that glp-1 inhibits sirt1-mediated foxo1 deacetylation . a : foxo1 acetylation and binding to sirt1 were evaluated by western blot after immunoprecipitation of foxo1 protein complexes . ins832/13 cells were incubated in serum - free medium in the presence or absence of glp-1 ( 10 nmol / l ) , with or without the sirt1 activators resveratrol ( 10 mol / l ) and h2o2 ( 25 mol / l ) for 45 min . b : densitometry analysis of five different experiments as shown in a. * p < 0.05 . c : foxo1 acetylation was evaluated as described in a after the indicated periods of time . sirt1 was immunoprecipitated from cross - linked chromatin extracted from ins832/13 cells treated with or without glp-1 for 45 min . eluted dna was pcr - amplified using oligonucleotides flanking the forkhead binding site in the rat foxa2 promoter . e : western blots showing the subcellular localization of sirt1 and foxo1 after 45-min incubation with either 10 nmol / l glp-1 ( glp ) or 10 mol / l resveratrol . chromatin immunoprecipitation assay was performed to assess whether sirt1 is recruited to the promoter region of foxo1 target genes . figure 1d shows that sirt1 was physically associated to a region of the foxa2 promoter encompassing the forkhead binding site ( 10 ) . this result indicates that sirt1 binding to the foxa2 promoter mirrors that of foxo1 , as we have previously demonstrated ( 10 ) . altogether , these findings indicate that sirt1 interacts with foxo1 and is recruited to the promoter of foxo1 target genes in a glp-1inhibitable manner . these data suggest a role for sirt1 in the transcriptional response to the glucoincretin hormone in -cells . we next performed subcellular fractionation to address whether glp-1 provokes sirt1 nuclear exclusion , as it does for foxo1 ( fig . our results show that sirt1 is predominantly nuclear in ins cells and that its subcellular localization was not altered by glp-1 treatment . conversely , glp-1 induced cytoplasmic translocation of foxo1 , as we have published previously ( 10 ) . we next sought to directly test whether glp-1 affects sirt1 activity using an in vitro deacetylase assay . figure 2a shows that glp-1 blunted sirt1 activity to the same extent as the sirt1 inhibitor nicotinamide . conversely , sirt1 activity was increased in resveratrol - treated cells . because sirt1 activity is dependent on nad , we measured changes in the nad - to - nadh ratio in response to glp-1 treatment in ins cells and isolated human islets . figure 2b shows that glp-1 decreased the nad - to - nadh ratio by 30% in ins cells , an action mimicked by the addition of 10% serum . conversely , resveratrol increased the nad - to - nadh ratio by 60% . both glp-1 and serum also elicited a reduction in the nad - to - nadh ratio in human islets ( fig . 2c ) , whereas the effect of resveratrol ( 25% ) did not reach statistical significance . the reduction in nad availability provides a possible mechanism by which glp-1 could acutely regulate sirt1 activity . to directly test this possibility , we supplemented the assay buffer with 450 mol / l nad ( fig . supplementation of nad suppressed glp-1 as well as serum inhibition of sirt1 activity . in an effort to unravel the mechanism by which glp-1 could alter the nad - to - nadh ratio , we investigated its action on the activity of nampt , the rate - limiting enzyme for the biosynthesis of nad , and on the cellular atp content . 2e ) , thereby revealing a novel mechanism by which glp-1 could decrease the nad - to - nadh ratio . fk-866 ( 10 nmol / l ) , a specific nampt pharmacological inhibitor , was used as a positive control . 2f ) , consistent with a recent publication by peyot et al . ( 28 ) . it is interesting that the addition of serum did not alter nampt activity but significantly increased the cellular atp concentration . a : cells were incubated in the presence or absence of 10 nmol / l glp-1 , 10 mmol / l nicotinamide ( nam ) , or 10 mol / l resveratrol ( res ) . proteins were extracted after 15 min to perform the in vitro sirt1 deacetylase assay using a fluorogenic sirt1 substrate . b and c : nad and nadh levels were determined in extracts from cells ( b ) or human islets ( c ) incubated in the presence or absence of 10 nmol / l glp-1 , 10% serum , or 10 mol / l resveratrol for 15 min . d : sirt1 activity was measured as described in a with the modification that the assay buffer was supplemented with 25 mol / l nad . e : nampt activity was measured by the conversion of c - labeled nicotinamide to [ c]nicotinamide mononucleotide . the specific nampt pharmacological inhibitor fk-866 ( 10 we next measured sirt1 expression by quantitative pcr after overnight incubation in serum - free medium in the absence or presence of glp-1 ( fig . our data show that glucose and serum deprivation , a condition mimicking calorie restriction , increased sirt1 expression by fivefold compared with complete growth medium in ins cells . 3b ) . both glp-1 and serum decreased sirt1 expression in rat islets as well , although the detected changes were more modest . thus , sirt1 expression was repressed by 30% in the presence of glp-1 and by 40% in response to serum . we assessed sirt1 protein levels by western blot to test whether alterations in sirt1 expression were translated into changes in protein levels . figure 3c and d shows that glp-1 decreased sirt1 protein levels by 70% in ins cells , an effect mimicked by serum . a and b : cells ( a ) and isolated rat islets ( b ) were incubated in serum - free medium in the absence or presence of glp-1 or left untreated in complete growth medium for 24 h. sirt1 expression levels were measured by quantitative pcr and normalized to actin . c : sirt1 protein levels were evaluated by western blot in ins cells treated as described in a and b. d : densitometry analysis of three different experiments as shown in a and b. * p < 0.05 . to test whether sirt1 inactivation mediates glp-1 action on -cell proliferation , we sought to perform sirt1 gain of function both in vitro and in vivo . ins832/13 cells were either transfected with wild - type sirt1 , a catalytically inactive sirt1 mutant , or with control gfp , and -cell replication was evaluated by brdu incorporation . transfection of the cells with sirt1 led to a fivefold increase in protein levels as compared with gfp ( fig . overexpression of wild - type sirt1 inhibited glp-1induced -cell proliferation but failed to significantly affect the effect of serum ( fig . overexpression of a catalytically inactive sirt1 mutant did not significantly alter -cell proliferation , indicating that the effects of sirt1 on -cell proliferation are dependent on its deacetylase activity . a : sirt1 protein levels were evaluated by western blot after transfection with wild - type ( wt ) or dominant - negative ( dn)-sirt1 . b : the effect of sirt1 overexpression on -cell proliferation was evaluated by brdu incorporation . ins832/13 cells were transfected with wt - sirt1 , dn - sirt1 , or control gfp for 24 h and then incubated in the presence or absence of glp-1 ( 10 nmol / l ) or 10% serum for an additional 24-h period . results represent mean  sem of three separate experiments carried out in triplicate . * p < 0.05 compared with the respective control . to demonstrate the implication of sirt1 in glp-1 action in vivo , we determined whether the effects of glp-1 on -cell mass were curtailed in transgenic mice with increased dosage of sirt1 ( sirbaco mice ) as previously described ( 27 ) . thus , mice were administered with either saline or the long - acting glp-1 analog exendin-4 ( 10 nmol / kg ) daily for 7 days . cross - sectional -cell areas were estimated by morphometry of pancreatic sections stained for insulin ( fig . exendin-4 administration increased the relative -cell area by 2.5-fold and stimulated -cell replication by 50% in wild - type animals . however , glp-1 effects on both -cell area and -cell replication were abolished in sirbaco mice . taken together , our results indicate that sirt1 gain of function prevents the mitogenic action of glp-1 and identify sirt1 as a negative regulator of -cell mass expansion . wild - type ( wt ) or sirbaco transgenic mice received daily injections of exendin-4 ( 10 nmol / kg body wt ) or saline for 7 days . pancreatic -cell mass and -cell replication were evaluated by morphometry and immunohistochemistry with anti - insulin and anti - ki67 antisera . a and b : the relative cross - sectional islet area ( a ) and the percentage of ki67-positive -cells ( b ) are shown . restoration and maintenance of -cell mass is a major goal of diabetes treatment ( 2931 ) . glp-1 , a potent antidiabetes medication ( 4 ) , enhances glucose - stimulated insulin secretion and promotes -cell mass expansion , at least in rodents . however , the molecular mechanism by which glp-1 exerts its action on -cell mass is not fully elucidated . we have previously demonstrated that the transcription factor foxo1 , an important regulator of -cell mass ( 1618 ) , is a prominent transcriptional effector of glp-1 action ( 10 ) . thus , glp-1 provoked nuclear exclusion of foxo1 to relieve a molecular brake on pdx1 and foxa2 expression . also , the effect of exendin-4 on -cell mass expansion was abolished in transgenic mice with -cell  specific foxo1 gain of function ( 10 ) . acetylation / deacetylation of foxo1 regulates its transcriptional activity ( 19 ) . on one hand , acetylation of foxo1 by cbp / p300 reduces its dna binding activity and increases its susceptibility to phosphorylation by akt ( 20 ) . on the other hand , acetylated foxo1 binds to the promyelocytic leukemia  associated protein pml and is targeted to specific nuclear subdomains ( pml nuclear bodies ) ( 32 ) . upon calorie restriction or oxidative stress , foxo1 is deacetylated by the nad - dependent sirtuin deacetylase sirt1 , which renders foxo1 immobile within the nuclear compartment and results in foxo1 transcriptional activity ( 26 ) . our findings show that glp-1 increases foxo1 acetylation via inhibition of the nad+-dependent sirt1 deacetylase . first , we show that glp-1 acutely decreases the cellular nad - to - nadh ratio , possibly via its hitherto uncharacterized action on nampt . second , glp-1 downregulates sirt1 expression , thereby providing a long - term mechanism by which chronic or prolonged exposure to glp-1 receptor agonists could also inhibit sirt1 . the fact that glp-1 failed to increase the intracellular atp content seemingly refutes the hypothesis that glp-1 could inhibit sirt1 via the stimulation of -cell glucose metabolism . however , because our assay did not take into account compartmentalization of atp production or breakdown , this possibility can not be completely ruled out . we also demonstrate that the effects of glp-1 and its long - acting analog exendin-4 on -cell mass were blunted in a pancreatic -cell line with increased dosage or sirt1 as well as in transgenic mice with sirt1 gain of function . our results are consistent with a model in which glp-1 inhibits sirt1-mediated foxo1 deacetylation to relieve the constraint on -cell proliferation and -cell mass expansion ( fig . thus far , the action of glp-1 agonists on -cell mass has not been demonstrated in humans in vivo . moreover , the reported increase in -cell mass in mice has been shown to be restricted after the initial burst of proliferation in the postnatal period ( 33,34 ) . however , a recent publication suggests that exendin-4 can stimulate -cell replication in human islet grafts ( 35 ) . proposed model . mmp , matrix metalloproteinase ; egf , epidermal growth factor ; egfr , epidermal growth factor receptor ; pi-3k , phosphatidylinositol-3 kinase . ( a high - quality color representation of this figure is available in the online issue . ) previous studies using genetically engineered mice with sirt1 gain ( 36 ) or loss of function ( 37 ) indicate that sirt1 could enhance insulin secretion , an effect mediated via reduction of ucp2 expression . these findings garnered much attention by identifying sirt1 , a molecule already scrutinized for its role in longevity , as a potential target for diabetes treatment . however , the action of sirt1 on insulin secretion is difficult to conciliate with its role in calorie restriction ( 38,39 ) . this discrepancy may be attributed to differences in the level and pattern of sirt1 expression between the various animal models . at first glance , our findings that the glucoincretin hormone glp-1 inhibits sirt1 may seem conflicting with the action of sirt1 on insulin secretion . however , the short - term effects of glp-1 on insulin secretion are not transcriptional in nature ( 40 ) . in conclusion our study describes a novel mechanism of action for the glucoincretin hormone and identifies for the first time sirt1 as a negative regulator of pancreatic -cell mass . thus , sirt1 could link -cell replication to the hormonal / nutritional status of the cells .