body and pancreas weight were recorded , and blood glucose ( glucometer elite , bayer , terrytown , nj ) , plasma insulin ( enzyme - linked immunosorbent assay ) , and glucagon ( radioimmunoassay ) levels ( joslin derc specialized assay core , boston , ma ) were measured before experiments .
glucose tolerance tests , acute glucose - stimulated insulin secretion ( gsis ) tests , and insulin tolerance tests were performed as previously reported ( 27 ) .
pancreata were harvested for immunohistochemical analyses and islet morphometry , and islets were isolated for rna extraction and in vitro gsis on batches of 15 size - matched islets as reported previously ( 28 ) .
pancreas sections were used to assess -cell mass and size as described previously ( 27 ) .
-cell proliferation was assessed in pancreas sections as described previously ( 28 ) , using anti - insulin ( guinea - pig antibody , dako , carpinteria , ca ) , anti - brdu ( rabbit polyclonal antibody , dako ) , or antiphosphohistone h3 antibodies ( rabbit polyclonal antibody , upstate biotechnology , lake placid , ny ) .
other antibodies used include -catenin ( rabbit polyclonal antibody , bd biosciences , oxford , u.k . ) , glucagon ( mouse monoclonal antibody , sigma - aldrich , st .
louis , mo ) , somatostatin ( rabbit polyclonal antibody , abcam , cambridge , u.k . ) , p21 ( mouse monoclonal antibody , dako ) , insulin ( mouse monoclonal antibody , biogenex , san ramon , ca ) , and dapi ( sigma - aldrich ) for nuclear staining .
quantitative real - time rt - pcr was performed on total rna samples extracted from islets and processed as described previously ( 28 ) .
antibodies used include cyclin d2 ( rabbit polyclonal antibody , santa cruz biotechnology inc . , santa cruz , ca ) , foxo1 ( rabbit polyclonal antibody , cell signaling , danvers , ma ) , pancreatic duodenal homeobox-1 ( pdx-1 ) ( gift from c.v .
wright , vanderbilt university ) , p27 ( mouse monoclonal antibody , bd biosciences ) , p21 ( mouse monoclonal antibody , dako ) , and tubulin ( mouse monoclonal antibody , abcam ) .
all data are presented as mean  sem and were analyzed using an anova or unpaired , two - tailed student t test as appropriate .
body and pancreas weight were recorded , and blood glucose ( glucometer elite , bayer , terrytown , nj ) , plasma insulin ( enzyme - linked immunosorbent assay ) , and glucagon ( radioimmunoassay ) levels ( joslin derc specialized assay core , boston , ma ) were measured before experiments .
glucose tolerance tests , acute glucose - stimulated insulin secretion ( gsis ) tests , and insulin tolerance tests were performed as previously reported ( 27 ) .
pancreata were harvested for immunohistochemical analyses and islet morphometry , and islets were isolated for rna extraction and in vitro gsis on batches of 15 size - matched islets as reported previously ( 28 ) .
pancreas sections were used to assess -cell mass and size as described previously ( 27 ) .
-cell proliferation was assessed in pancreas sections as described previously ( 28 ) , using anti - insulin ( guinea - pig antibody , dako , carpinteria , ca ) , anti - brdu ( rabbit polyclonal antibody , dako ) , or antiphosphohistone h3 antibodies ( rabbit polyclonal antibody , upstate biotechnology , lake placid , ny ) .
other antibodies used include -catenin ( rabbit polyclonal antibody , bd biosciences , oxford , u.k . ) , glucagon ( mouse monoclonal antibody , sigma - aldrich , st .
louis , mo ) , somatostatin ( rabbit polyclonal antibody , abcam , cambridge , u.k . ) , p21 ( mouse monoclonal antibody , dako ) , insulin ( mouse monoclonal antibody , biogenex , san ramon , ca ) , and dapi ( sigma - aldrich ) for nuclear staining .
quantitative real - time rt - pcr was performed on total rna samples extracted from islets and processed as described previously ( 28 ) .
antibodies used include cyclin d2 ( rabbit polyclonal antibody , santa cruz biotechnology inc . , santa cruz , ca ) , foxo1 ( rabbit polyclonal antibody , cell signaling , danvers , ma ) , pancreatic duodenal homeobox-1 ( pdx-1 ) ( gift from c.v .
wright , vanderbilt university ) , p27 ( mouse monoclonal antibody , bd biosciences ) , p21 ( mouse monoclonal antibody , dako ) , and tubulin ( mouse monoclonal antibody , abcam ) .
all data are presented as mean  sem and were analyzed using an anova or unpaired , two - tailed student t test as appropriate .
we compared the phenotype of mice at 3 , 10 , and 1214 months of age .
the phenotype of p44tg mice becomes evident at 3 months , and the animals begin to succumb at 14 months of age ( 22 ) .
p44tg mice were lighter than their nontransgenic counterparts at all ages , as shown for 3 months of age in fig .
the animals had already developed hypoinsulinemia at this age , which persisted throughout life ( fig .
1b and 2c ) , suggesting overt diabetes that could contribute , in part , to death at this age .
in contrast , all controls exhibited fed blood glucose levels < 150 mg / dl at 12 months of age ( fig .
these data indicate that increased dosage of 40p53 leads to hypoinsulinemia and glucose intolerance as early as 3 months and worsens with age .
body weight ( a ) , blood glucose ( b ) , plasma insulin ( c ) , and plasma glucagon ( d ) levels were measured at 3 , 10 , and 1214 months of age from random - fed control (  ) and p44tg (  ) ( n = 59 ) mice . * p < 0.05 for p44tg vs. control mice . # p < 0.05 for control mice vs. different ages .
glucose tolerance tests performed at 2 months ( a ) , 35 months ( b ) , and 1014 months ( c ) of age ( n = 68 ) .
blood glucose was measured at 0 , 10 , 20 , 30 , 60 , and 120 min after intraperitoneal injection of glucose .
insulin tolerance tests were performed in each group at 2 months ( d ) , 34 months ( e ) , and 1516 months ( f ) of age ( n = 68 ) .
glucose was measured at 0 , 15 , 30 , 45 , and 60 min after intraperitoneal injection of insulin .
ip , intraperitoneal ; ipgtt , intraperitoneal glucose tolerance test ; ipitt , intraperitoneal insulin tolerance test .
although both pancreas weight and number of islets exhibited an age - dependent increase in control mice , the total number of islets was less in p44tg mice even when normalized to total pancreas weight ( fig .
1 ) . compared with 3 months of age , the differences became more marked at 1214 months .
the reduced pancreatic weight and islet number in p44tg mice were associated with a dramatic decrease in -cell mass ( fig .
-cell mass was significantly lower in the p44tg mice and ranged between 11% ( young ) and 14% ( old ) in the age - matched control animals .
quantification of -cell size revealed a 30% decrease in the p44tg mice compared with controls at 3 months of age but showed the opposite in the older p44tg mice ( fig .
this represented a twofold increase in -cell size as the p44tg mice aged from 3 to 12 months . to explore the pathways that regulate -cell size , we considered previous reports that proteins in the insulin / igf-1 signaling pathways regulate -cell size via akt / mtor / s6k1 ( 1416 ) . quantitative real - time rt - pcr for these molecules did not reveal significant differences between groups at 3 months of age except for a decrease in insulin receptor ( ir ) expression in p44tg islets ( fig .
3e ) . however , in islets from older p44tg mice , we observed increased gene expression of ir , irs1 ( ir substrate-1 ) , and especially akt1 , mtor , and s6k1 , suggesting an attempt at compensation by increasing the -cell size in the older mutant mice .
this was not observed in the liver or white adipose tissue of p44tg mice ( supplementary fig .
a : pancreatic weight expressed as a percentage of the total body weight ( n = 3 ) .
c and d : -cell size was assessed by coimmunostaining for -catenin ( green ) and insulin ( red ) with dapi ( blue ) in pancreas sections from control ( n = 5 ) and p44tg ( n = 5 ) mice at 3 and 1214 months of age as described in research design and methods .
a representative islet for each group at magnification 40 is presented with the quantification of relative -cell size ( mean  sem from n 200 cells counted per mouse ) .
e : real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice . results are normalized to tata - binding protein ( tbp ) and expressed relative to control mice .
* p < 0.05 for p44tg vs. control mice . # p < 0.05 for 3- vs. 12- to 14-month - old control mice .
( a high - quality digital representation of this figure is available in the online issue . ) examination of islet morphology by immunohistochemistry ( fig .
4a ) revealed the typical pattern of a core of insulin - containing cells surrounded by non-cells in islets from controls and in a majority of islets from p44tg mice at 3 months of age ( fig .
however , 35% of islets in the normoglycemic and hypoinsulinemic p44tg groups exhibited infiltration of - and -cells within the islet core and a relative decrease in the number of -cells ( quantified in fig .
these abnormal features , which were not observed at 2 months of age ( supplementary fig .
2 ) , were evident in 85% of the islets by 12 months of age in the p44tg group ( fig . 4a , bottom ) . in parallel ,
4c ) revealed a significant increase in somatostatin gene expression that was consistent with a relative increase in -cells in p44tg mice ( fig .
a significant increase in the expression of insulin and glut 2 in 3-month - old transgenic mice suggested a compensatory effect at this younger age ( fig .
4c , top ) that was not evident in the older hyperglycemic group ( fig .
4c , bottom ) . because p44tg mice exhibit hypoinsulinemia and reduced -cell numbers , we evaluated -cell secretory function in vivo ( fig .
2b ) , the gsis in isolated islets in vitro was not different between groups ( fig .
reduction in insulin - positive cells and increase in somatostatin - positive cells in islets from p44tg mice .
a : triple coimmunostaining for insulin ( blue ) , somatostatin ( green ) , and glucagon ( red ) on pancreas sections from control or p44tg mice at 3 and 1214 months of age as described in research design and methods ( n = 5 ) .
b : the number of insulin - positive cells was counted in at least 10 islets randomly selected from pancreas sections from each mouse ( n = 3 mice at each age per group ) .
c : real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
insulin was measured in plasma samples extracted from blood collected at 0 , 2 , and 5 min after intraperitoneal injection of glucose ( 3 g / kg body wt ) from 45-month - old control and p44tg mice .
islets isolated from 45-month - old control and p44tg mice were incubated for 1 h with 3 or 16.7 mmol / l glucose as described in research design and methods .
bar graph depicts data obtained from three to five individual batches of size - matched islets from at least three independent mice per group .
* p < 0.05 for p44tg vs. control mice . # p < 0.05 for 3- vs. 12- to 14-month - old control mice .
( a high - quality digital representation of this figure is available in the online issue . )
next , we determined whether the decrease in -cell mass in p44tg mice ( fig .
coimmunostaining of pancreas sections with antibodies against insulin and either one of two proliferation markers showed a decrease in the proliferation of -cells with age in control islets ( fig .
brdu labeling demonstrated a virtual absence of replicating -cells in young and old p44tg mice compared with controls ( fig .
5a ) . a decrease in phh3 immunoreactive -cells at 3 and 1214 months of age in p44tg mice confirmed reduced number of cells in the m or late g2 phases of the cell cycle ( fig .
, no significant difference was evident in -cell replication between the two groups ( supplementary fig .
-cell proliferation was assessed by coimmunostaining for brdu ( a , green ) and phh3 ( b , green ) with insulin ( red ) and dapi ( blue ) in pancreas sections from control and p44tg mice at 3 and 1214 months of age as described in research design and methods . a representative islet for each group at magnification 40 is presented ( n = 5 ) .
p < 0.05 for p44tg vs. control mice . # p < 0.05 for 3- vs. 12- to 14-month - old control mice .
( a high - quality digital representation of this figure is available in the online issue . )
we next evaluated the effects of neogenesis ( 30 ) and apoptosis ( 31 ) in the maintenance of -cell mass .
single and clusters ( 8 ) of insulin+ cells ( markers of neogenesis ) were virtually absent in pancreatic ducts in 3-month - old control and p44tg mice .
although some insulin+ cells / clusters were observed in 12- to 14-month - old mice , no significant difference was evident between groups ( number / mm pancreas : 0.3  0 , control vs. 0.3  0.2 , p44tg ; p = ns , n = 3 ) .
similarly , no significant differences were observed in insulin+ cells in extraductal pancreatic tissue ( 3 month ; 14  0.3 , control vs. 12.8  3.4 , p44tg ; 1214 month ; 8.3  0.2 , control vs. 10.2  2.4 , p44tg ; p = ns ; n = 3 ) , suggesting that neogenesis is not altered significantly when 40p53 is misexpressed .
although p53 has been studied in the context of apoptosis in multiple tissues , it has not been causally involved in -cell death during diabetes ( 32 ) .
nevertheless , we performed the transferase - mediated dutp nick - end labeling ( tunel ) assay and found no significant differences in tunel+ -cells between groups ( supplementary fig .
these data provide strong evidence that the misexpression of 40p53 led to a marked reduction of -cell proliferation , consistent with reports for other cell types ( 22,24 ) .
p53 regulates the g1 to s phase progression through cell cycle inhibitors such as p21 , but recent reports indicate that p53 also targets integral cell cycle proteins , such as cyclin e and cdk4 ( 18 ) .
we observed a significant decrease in the gene expression levels of cyclin d2 , cdk2 , and cyclin e1 , and a significant increase in the level of cyclin e2 in 3-month - old p44tg mice ( fig .
only cyclin e1 gene expression remained low ; at the same time , however , cdk4 levels became significantly increased .
the decrease in cyclin d2 gene expression at 3 months of age correlated with lower protein expression in isolated islets ( fig .
in old mice , despite normal levels of gene expression , cyclin d2 protein level was significantly decreased ( fig .
a : real time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for cyclin d2 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . * p < 0.05 for p44tg vs. control mice .
we next examined the expression of foxo1 , which regulates -cell proliferation ( 33 ) , and two of its target proteins , pdx-1 ( 34 ) and p27 ( 35 ) .
examination of gene and protein expression in whole islets revealed that foxo1 expression was significantly lower at 3 months but unchanged at 10 months in p44tg mice ( fig .
this decrease was less pronounced in old mice and correlated with fewer -cells in islets from the transgenic mice ( fig .
4b ) . p27 gene and protein expression remained unchanged ( fig . 7a and b ) .
these data indicate that decreased -cell mass and proliferation observed in p44tg mice ( figs .
a : real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for foxo1 , pdx-1 , and p27 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . *
. finally , we evaluated the gene expression of p53 and its protein targets ( 19 ) . to measure the level of transcription of both p53 ( endogenous ) and 40p53 ( endogenous and transgene ) genes , we designed two sets of primers for quantitative rt - pcr because the use of specific primers to distinguish both genes was not possible .
np53 primers targeted the 5 end of the p53 transcript and amplified transcripts derived from the endogenous p53 gene only , whereas cp53 primers targeted sequences at the 3 end of rna and amplified transcripts derived from both the endogenous p53 gene and the transgene .
we also measured the expression of several well - known p53 target genes ( 19 ) , such as p21 , 143 - 3 , gadd45 , bax , mdm2 , pten , and the igf-1 receptor ( igf-1r ) .
we observed a significant decrease in endogenous p53 gene expression ( np53 primers ) that correlated with reduced gadd45 gene in islets from 3-month - old p44tg mice ( fig .
8a , top ) . in islets from 12-month - old p44tg mice , we observed an increase in 40p53 expression ( cp53 primers ) that was associated with an enhanced expression of p21 and mdm2 ( fig .
although bax gene expression was significantly elevated in p44tg islets at both ages , we did not observe significance differences between groups in the tunel assay ( supplementary fig .
the increase in gene expression of igf-1r that was evident in young p44tg islets did not persist in older mice , and we did not observe alterations in pten gene expression at either age ( fig .
however , we did find an increase in p21 protein by western blot analysis of p44tg islets ( fig .
thus , the strong inhibition of -cell proliferation observed in p44tg islet cells correlated with a significant increase in p21 , the main target of p53 in cell cycle regulation . increased p21 expression in p44tg islets .
a : real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for p21 and quantification normalized to actin in islets from control and p44tg at 3 and 10 months of age . *
c : quantification of p21-positive -cell nuclei in pancreas sections from control and p44tg mice at 3 and 1214 months of age ( n = 5 ) .
* p < 0.05 for p44tg vs. control mice ( at least 810 islets from five mice per group ) .
we compared the phenotype of mice at 3 , 10 , and 1214 months of age .
the phenotype of p44tg mice becomes evident at 3 months , and the animals begin to succumb at 14 months of age ( 22 ) .
p44tg mice were lighter than their nontransgenic counterparts at all ages , as shown for 3 months of age in fig .
the animals had already developed hypoinsulinemia at this age , which persisted throughout life ( fig .
1b and 2c ) , suggesting overt diabetes that could contribute , in part , to death at this age .
in contrast , all controls exhibited fed blood glucose levels < 150 mg / dl at 12 months of age ( fig .
these data indicate that increased dosage of 40p53 leads to hypoinsulinemia and glucose intolerance as early as 3 months and worsens with age .
body weight ( a ) , blood glucose ( b ) , plasma insulin ( c ) , and plasma glucagon ( d ) levels were measured at 3 , 10 , and 1214 months of age from random - fed control (  ) and p44tg (  ) ( n = 59 ) mice . * p < 0.05 for p44tg vs. control mice . # p < 0.05 for control mice vs. different ages .
glucose tolerance tests performed at 2 months ( a ) , 35 months ( b ) , and 1014 months ( c ) of age ( n = 68 ) .
blood glucose was measured at 0 , 10 , 20 , 30 , 60 , and 120 min after intraperitoneal injection of glucose .
insulin tolerance tests were performed in each group at 2 months ( d ) , 34 months ( e ) , and 1516 months ( f ) of age ( n = 68 ) .
glucose was measured at 0 , 15 , 30 , 45 , and 60 min after intraperitoneal injection of insulin .
ip , intraperitoneal ; ipgtt , intraperitoneal glucose tolerance test ; ipitt , intraperitoneal insulin tolerance test .
although both pancreas weight and number of islets exhibited an age - dependent increase in control mice , the total number of islets was less in p44tg mice even when normalized to total pancreas weight ( fig .
1 ) . compared with 3 months of age , the differences became more marked at 1214 months . the reduced pancreatic weight and islet number in p44tg mice were associated with a dramatic decrease in -cell mass ( fig .
-cell mass was significantly lower in the p44tg mice and ranged between 11% ( young ) and 14% ( old ) in the age - matched control animals .
quantification of -cell size revealed a 30% decrease in the p44tg mice compared with controls at 3 months of age but showed the opposite in the older p44tg mice ( fig .
this represented a twofold increase in -cell size as the p44tg mice aged from 3 to 12 months . to explore the pathways that regulate -cell size
, we considered previous reports that proteins in the insulin / igf-1 signaling pathways regulate -cell size via akt / mtor / s6k1 ( 1416 ) . quantitative real - time rt - pcr for these molecules did not reveal significant differences between groups at 3 months of age except for a decrease in insulin receptor ( ir ) expression in p44tg islets ( fig .
3e ) . however , in islets from older p44tg mice , we observed increased gene expression of ir , irs1 ( ir substrate-1 ) , and especially akt1 , mtor , and s6k1 , suggesting an attempt at compensation by increasing the -cell size in the older mutant mice .
this was not observed in the liver or white adipose tissue of p44tg mice ( supplementary fig .
a : pancreatic weight expressed as a percentage of the total body weight ( n = 3 ) .
c and d : -cell size was assessed by coimmunostaining for -catenin ( green ) and insulin ( red ) with dapi ( blue ) in pancreas sections from control ( n = 5 ) and p44tg ( n = 5 ) mice at 3 and 1214 months of age as described in research design and methods .
a representative islet for each group at magnification 40 is presented with the quantification of relative -cell size ( mean  sem from n 200 cells counted per mouse ) .
e : real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
results are normalized to tata - binding protein ( tbp ) and expressed relative to control mice . * p < 0.05 for p44tg vs. control mice
. # p < 0.05 for 3- vs. 12- to 14-month - old control mice . 
( a high - quality digital representation of this figure is available in the online issue . )
4a ) revealed the typical pattern of a core of insulin - containing cells surrounded by non-cells in islets from controls and in a majority of islets from p44tg mice at 3 months of age ( fig .
4a , top ) . however , 35% of islets in the normoglycemic and hypoinsulinemic p44tg groups exhibited infiltration of - and -cells within the islet core and a relative decrease in the number of -cells ( quantified in fig .
these abnormal features , which were not observed at 2 months of age ( supplementary fig .
2 ) , were evident in 85% of the islets by 12 months of age in the p44tg group ( fig .
4c ) revealed a significant increase in somatostatin gene expression that was consistent with a relative increase in -cells in p44tg mice ( fig .
4a ) , whereas glucagon gene expression ( fig . 4c ) and circulating glucagon levels ( fig .
a significant increase in the expression of insulin and glut 2 in 3-month - old transgenic mice suggested a compensatory effect at this younger age ( fig .
4c , top ) that was not evident in the older hyperglycemic group ( fig .
4c , bottom ) . because p44tg mice exhibit hypoinsulinemia and reduced -cell numbers , we evaluated -cell secretory function in vivo ( fig .
2b ) , the gsis in isolated islets in vitro was not different between groups ( fig .
reduction in insulin - positive cells and increase in somatostatin - positive cells in islets from p44tg mice .
a : triple coimmunostaining for insulin ( blue ) , somatostatin ( green ) , and glucagon ( red ) on pancreas sections from control or p44tg mice at 3 and 1214 months of age as described in research design and methods ( n = 5 ) .
b : the number of insulin - positive cells was counted in at least 10 islets randomly selected from pancreas sections from each mouse ( n = 3 mice at each age per group ) .
c : real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice . results are normalized to tbp and expressed relative to controls .
insulin was measured in plasma samples extracted from blood collected at 0 , 2 , and 5 min after intraperitoneal injection of glucose ( 3 g / kg body wt ) from 45-month - old control and p44tg mice .
islets isolated from 45-month - old control and p44tg mice were incubated for 1 h with 3 or 16.7 mmol
bar graph depicts data obtained from three to five individual batches of size - matched islets from at least three independent mice per group . *
. # p < 0.05 for 3- vs. 12- to 14-month - old control mice .
( a high - quality digital representation of this figure is available in the online issue . )
next , we determined whether the decrease in -cell mass in p44tg mice ( fig .
coimmunostaining of pancreas sections with antibodies against insulin and either one of two proliferation markers showed a decrease in the proliferation of -cells with age in control islets ( fig .
brdu labeling demonstrated a virtual absence of replicating -cells in young and old p44tg mice compared with controls ( fig .
5a ) . a decrease in phh3 immunoreactive -cells at 3 and 1214 months of age in p44tg mice confirmed reduced number of cells in the m or late g2 phases of the cell cycle ( fig .
, no significant difference was evident in -cell replication between the two groups ( supplementary fig .
-cell proliferation was assessed by coimmunostaining for brdu ( a , green ) and phh3 ( b , green ) with insulin ( red ) and dapi ( blue ) in pancreas sections from control and p44tg mice at 3 and 1214 months of age as described in research design and methods .
a representative islet for each group at magnification 40 is presented ( n = 5 ) .
quantification is shown on the right . * p < 0.05 for p44tg vs. control mice
. # p < 0.05 for 3- vs. 12- to 14-month - old control mice .
( a high - quality digital representation of this figure is available in the online issue . )
we next evaluated the effects of neogenesis ( 30 ) and apoptosis ( 31 ) in the maintenance of -cell mass .
single and clusters ( 8 ) of insulin+ cells ( markers of neogenesis ) were virtually absent in pancreatic ducts in 3-month - old control and p44tg mice .
although some insulin+ cells / clusters were observed in 12- to 14-month - old mice , no significant difference was evident between groups ( number / mm pancreas : 0.3  0 , control vs. 0.3  0.2 , p44tg ; p = ns , n = 3 ) .
similarly , no significant differences were observed in insulin+ cells in extraductal pancreatic tissue ( 3 month ; 14  0.3 , control vs. 12.8  3.4 , p44tg ; 1214 month ; 8.3  0.2 , control vs. 10.2  2.4 , p44tg ; p = ns ; n = 3 ) , suggesting that neogenesis is not altered significantly when 40p53 is misexpressed .
although p53 has been studied in the context of apoptosis in multiple tissues , it has not been causally involved in -cell death during diabetes ( 32 ) .
nevertheless , we performed the transferase - mediated dutp nick - end labeling ( tunel ) assay and found no significant differences in tunel+ -cells between groups ( supplementary fig .
these data provide strong evidence that the misexpression of 40p53 led to a marked reduction of -cell proliferation , consistent with reports for other cell types ( 22,24 ) .
p53 regulates the g1 to s phase progression through cell cycle inhibitors such as p21 , but recent reports indicate that p53 also targets integral cell cycle proteins , such as cyclin e and cdk4 ( 18 ) .
we observed a significant decrease in the gene expression levels of cyclin d2 , cdk2 , and cyclin e1 , and a significant increase in the level of cyclin e2 in 3-month - old p44tg mice ( fig .
only cyclin e1 gene expression remained low ; at the same time , however , cdk4 levels became significantly increased .
the decrease in cyclin d2 gene expression at 3 months of age correlated with lower protein expression in isolated islets ( fig .
in old mice , despite normal levels of gene expression , cyclin d2 protein level was significantly decreased ( fig .
a : real time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for cyclin d2 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . *
we next examined the expression of foxo1 , which regulates -cell proliferation ( 33 ) , and two of its target proteins , pdx-1 ( 34 ) and p27 ( 35 ) .
examination of gene and protein expression in whole islets revealed that foxo1 expression was significantly lower at 3 months but unchanged at 10 months in p44tg mice ( fig .
this decrease was less pronounced in old mice and correlated with fewer -cells in islets from the transgenic mice ( fig .
4b ) . p27 gene and protein expression remained unchanged ( fig . 7a and b ) .
these data indicate that decreased -cell mass and proliferation observed in p44tg mice ( figs .
a : real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for foxo1 , pdx-1 , and p27 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . *
finally , we evaluated the gene expression of p53 and its protein targets ( 19 ) . to measure the level of transcription of both p53 ( endogenous ) and 40p53 ( endogenous and transgene ) genes , we designed two sets of primers for quantitative rt - pcr because the use of specific primers to distinguish both genes was not possible .
np53 primers targeted the 5 end of the p53 transcript and amplified transcripts derived from the endogenous p53 gene only , whereas cp53 primers targeted sequences at the 3 end of rna and amplified transcripts derived from both the endogenous p53 gene and the transgene .
we also measured the expression of several well - known p53 target genes ( 19 ) , such as p21 , 143 - 3 , gadd45 , bax , mdm2 , pten , and the igf-1 receptor ( igf-1r ) .
we observed a significant decrease in endogenous p53 gene expression ( np53 primers ) that correlated with reduced gadd45 gene in islets from 3-month - old p44tg mice ( fig .
8a , top ) . in islets from 12-month - old p44tg mice , we observed an increase in 40p53 expression ( cp53 primers ) that was associated with an enhanced expression of p21 and mdm2 ( fig .
although bax gene expression was significantly elevated in p44tg islets at both ages , we did not observe significance differences between groups in the tunel assay ( supplementary fig .
the increase in gene expression of igf-1r that was evident in young p44tg islets did not persist in older mice , and we did not observe alterations in pten gene expression at either age ( fig .
however , we did find an increase in p21 protein by western blot analysis of p44tg islets ( fig .
thus , the strong inhibition of -cell proliferation observed in p44tg islet cells correlated with a significant increase in p21 , the main target of p53 in cell cycle regulation . increased p21 expression in p44tg islets .
a : real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control ( n = 38 ) and p44tg ( n = 4 ) mice .
b : western blotting for p21 and quantification normalized to actin in islets from control and p44tg at 3 and 10 months of age . *
c : quantification of p21-positive -cell nuclei in pancreas sections from control and p44tg mice at 3 and 1214 months of age ( n = 5 ) .
* p < 0.05 for p44tg vs. control mice ( at least 810 islets from five mice per group ) .
by using p44tg mice with an ectopic p53 gene that encodes 40p53 ( 36 ) , we have identified a role for the tumor suppressor p53 in the maintenance of normal -cell mass and glucose homeostasis .
although 40p53 has virtually no autonomous transcriptional activity , it can interact with full - length p53 to form tetramers that bind dna and activate or suppress target gene expression ( 36,37 ) .
expression of ectopic 40p53 in the mouse alters the balance between the full - length and short isoforms that normally exists and hyperactivates p53 ( 22,36,38 ) .
this increased activity of p53 can account for the higher levels of p21 transcripts , which we observed in -cells from 1012-month - old p44tg mice .
the expression of 40p53 in p44tg mice can also account for the higher level of mrna encoding the igf-1r , whose transcription is differentially regulated by wild - type and mutant p53 ( 39 ) .
an increased gene expression of igf-1r in liver and white adipose tissue from p44tg mice ( supplementary fig .
similar to embryonic fibroblasts derived from p44tg mice ( 22 ) , islets from young p44tg mice expressed significantly high levels of igf-1r , indicating impaired trans - suppression activity .
both the increased trans - activation of p21 in old islets and the decreased trans - suppression of the igf-1r in the young can be explained by differences in expression of the transgene encoding 40p53 with age , as detected by primers that amplify sequences encoding the cooh - terminal domain of the p53 protein .
younger p44tg mice expressed control levels of 40p53 and slightly lower levels of p53 , whereas older mice expressed normal levels of p53 and higher levels of 40p53 .
biochemical experiments have demonstrated that the stability and activity of the p53 tetramer are exquisitely sensitive to the dose of 40p53 , with low doses of 40p53 activating and high doses inactivating p53 function ( 40,41 ) .
the differential effects of 40p53 on p53-dependent trans - activation and trans - repression with age could also explain how p21 levels increase in both age groups , but by different mechanisms .
in old mice , the increase would be due to a direct effect of p53 on the p21 promoter , leading to increased transcription of the p21 gene .
in young mice , on the other hand , increased p21 could be an indirect effect of higher igf-1r expression and activation of igf signaling due to impaired igf-1r trans - repression .
stimulation of the igf-1 signal transduction pathway can increase p21 ( 42 ) , and further work is necessary to delineate the link among 40p53/p53 , p21 , and igf-1r levels in -cell proliferation .
-cell proliferation and glucose tolerance were impaired in 3-month - old p44tg animals and worsened to overt diabetes as the animals aged .
although random - fed blood glucose levels were normal in the transgenic mice at 3 and 10 months , the mutants displayed clear intolerance in response to a glucose challenge indicating deficiency in functional -cells .
for example , sreenan et al . ( 43 ) report a reduced -cell mass before the onset of diabetes in the nonobese diabetic mouse , and tavana et al .
( 44 ) describe the phenotypes of 1-month - old mice doubly mutant for p53 and nonhomologous end - joining deficiency that exhibit an 50% decrease in -cell mass and yet manifest blood glucose levels that are not significantly different from controls .
further , obese and nondiabetic humans have been reported to express a wide range of -cell mass that is adequate to maintain euglycemia up to a specific threshold , and crossing the threshold correlates with fasting hyperglycemia and overt diabetes ( 45 ) . in our study , it is possible that p44tg mice that are significantly leaner than controls at all ages only cross this  threshold  at 12 months of age when they begin to exhibit overt hyperglycemia .
thus , our data suggest that the balance between full - length and 40p53 isoforms plays a critical role in the maintenance of -cell proliferation and glucose homeostasis , and may be important in regulating the cell cycle during aging .
further support for a role for p53 in glucose homeostasis is evident from recent studies .
( 46 ) reported a role for adipose p53 in the regulation of insulin resistance . in our study , circulating proinflammatory cytokines , which are potential effectors in insulin resistance associated with diabetes , showed no significant differences between groups ( supplementary fig .
the cytokine expression profile in liver and white adipose tissue was also similar between groups .
these data , along with the normal insulin sensitivity in the global p44tg mice , indicate that the glucose intolerance is due to intrinsic effects of p53 on -cell mass rather than to secondary effects of transgene expression in other insulin sensitive tissues . indeed ,
( 44 ) reported that dna double - strand breaks combined with an absence of p53-dependent apoptosis in mice leads to reduced -cell replication and severe age - dependent diabetes , supporting our hypothesis that p53 plays a role in the regulation of -cell proliferation .
although the mechanisms that regulate the dynamics of -cell turnover during aging are still being unraveled , several studies including our own support a role for replication as the major mechanism that underlies the compensatory growth response to insulin resistance ( 15,47 ) .
two proteins that are important for this compensatory growth response are cyclin d2 ( 5,11,12 ) and pdx-1 , the pancreatic duodenal homeobox domain protein ( 3 ) . the lower expression of cyclin d2 in islets would potentially exacerbate the loss of -cells and lead to glucose intolerance and overt diabetes . in this study , the expression of pdx-1 was also altered in p44tg -cells .
although the gene expression of pdx-1 was increased in both young and old p44tg mice , the protein expression in islets was low , suggesting compensatory effects in gene expression or more dominant regulation of the transcription factor at the protein level .
in old p44tg mice , however , there were fewer -cell nuclei in which pdx-1 could be detected .
immunohistochemistry ( data not shown ) also revealed that the localization of pdx-1 correlated inversely with that of foxo1 , a transcription factor that regulates expression of this homeodomain protein ( 34 ) . because foxo1 transactivates cell cycle inhibitors ( p21 and p27 ) ( 35 ) and represses cell cycle activators ( cyclin d1 and d2 ) , nuclear localization of foxo1 could have a major impact on cell cycle progression in aging -cells .
it is also possible that foxo1 and p53 interact with deacetylase sirtuin 1 and function in a cooperative manner in an aging environment ( 48 ) .
thus , the alterations in both pdx-1 and cyclin d2 , in our model of accelerated aging , underscore the concept that these two proteins are involved in -cell replication during aging .
two cdkis , namely , p16 and p19 , have been reported to be involved in -cell regenerative failure in diabetes ( 49 ) .
our observations of enhanced expression of p16 and p19 genes confirm the premature aging phenotype of p44tg mice ( supplementary fig .
however , the regulatory link between p53 and the two aging markers in the maintenance or amplification of the phenotype requires further investigation .
( 22 ) previously described that young p44tg mice displayed signs of aging in bone .
further , the osteoblast - secreted molecule , osteocalcin , has recently been reported to modulate insulin secretion and insulin gene expression that correlated with expression of creb and neurod genes ( 50 ) . in our study
, we observed a mild but significant decrease in osteocalcin levels only in old p44tg mice ; however , gene expression of insulin , creb , or neurod was unaltered ( supplementary fig . 7 ) .
finally , the significance of the presence of p53 in non-cell fractions obtained from dispersed islet cells ( 29 ) remains unclear .
although the relative increase in -cells in p44tg islets was not associated with an increase in glucagon gene expression or circulating glucagon levels , we observed a significant increase in the number of somatostatin - secreting -cells and gene expression in the mutant islets .
the presence of a p53 response element in the somatostatin promoter ( genomatix ) warrants further studies to evaluate whether the increase in somatostatin actually decreases insulin secretion or limits -cell growth in a paracrine manner in the p44tg mice . in summary , increased dosage of 40p53 in mice promotes hypoinsulinemia and glucose intolerance , ultimately leading to overt diabetes with age and early death .
the suppression of -cell proliferation secondary to changes in the expression of p21 , cyclin d2 , and pdx-1 in this mouse model of accelerated aging implicates impaired p53 function in the development of type 2 diabetes in the elderly .
 objectiveinvestigating the dynamics of pancreatic -cell mass is critical for developing strategies to treat both type 1 and type 2 diabetes . 
 p53 , a key regulator of the cell cycle and apoptosis , has mostly been a focus of investigation as a tumor suppressor . 
 although p53 alternative transcripts can modulate p53 activity , their functions are not fully understood . 
 we hypothesized that -cell proliferation and glucose homeostasis were controlled by 40p53 , a p53 isoform lacking the transactivation domain of the full - length protein that modulates total p53 activity and regulates organ size and life span in mice.research design and methodswe phenotyped metabolic parameters in 40p53 transgenic ( p44tg ) mice and used quantitative rt - pcr , western blotting , and immunohistochemistry to examine -cell proliferation.resultstransgenic mice with an ectopic p53 gene encoding 40p53 developed hypoinsulinemia and glucose intolerance by 3 months of age , which worsened in older mice and led to overt diabetes and premature death from 14 months of age . 
 consistent with a dramatic decrease in -cell mass and reduced -cell proliferation , lower expression of cyclin d2 and pancreatic duodenal homeobox-1 , two key regulators of proliferation , was observed , whereas expression of the cell cycle inhibitor p21 , a p53 target gene , was increased.conclusionsthese data indicate a significant and novel role for 40p53 in -cell proliferation with implications for the development of age - dependent diabetes . 