male homozygous db / db and age- or sex - matched heterozygous littermates db/+ were obtained from the jackson laboratory ( bar harbor , mi ) .
male balb / c mice were purchased from charles river laboratories ( wilmington , ma ) .
mice were housed in standard shoebox cages supplied with standard rat diet and water ad libitum in a pathogen - free facility with a 12-h light / dark cycle . for lps treatment ,
male balb / c mice ( 25 g ) were injected intraperitoneally with lps at the dose of 4 mg / kg body weight for 2 consecutive days , as described ( 35 ) . to isolate peritoneal macrophages ,
mice were killed and peritoneal lavage fluid was collected by washing the peritoneum with 5-ml ice - cold hank 's balanced salt solution ( hbss ; gibco , carlsbad , ca ) .
subsequent preparation of peritoneal macrophages was according to the protocol as described ( 35 ) .
all procedures were approved by the institutional animal care and usage committee of children 's hospital of pittsburgh .
other methods including the statistics are described in the supplemental materials available in an online appendix at http://diabetes.diabetesjournals.org/cgi/full/db09-0232/dc1 .
aberrant proinflammatory cytokine production is intertwined with insulin resistance in obesity and type 2 diabetes . to decipher the molecular basis that links proinflammatory cytokine production to insulin resistance , we determined the effect of foxo1 on cytokine production in macrophages , a cell type that plays a pivotal role in mediating whole - body insulin resistance in the face of low - grade inflammation .
macrophage raw264.7 cells were transduced with adv - foxo1 or adv - null vector at a predefined moi ( 200 pfu / cell ) , followed by the determination of cytokine concentrations in conditioned medium . as shown in fig .
1 , transduction of raw264.7 cells by foxo1 vector resulted in a significant induction of il-1 and il-2 production without alterations in the production of other cytokines including il-4 , il-5 , il-6 , il-12 , ifn- , tnf- , and granulocyte monocyte colony - stimulating factor .
in contrast , il-10 expression was significantly downregulated in response to foxo1 production in raw264.7 cells . in the absence of lps ,
the expression of proinflammatory cytokines remained at low basal levels independently of foxo1 action ( supplemental table s1 ) .
these data underscore the importance of foxo1 in regulating cytokine production in lps - activated macrophages . because of the close association between abnormal production of il-1 in macrophages and the pathogenesis of insulin resistance , we focused on the delineation of the molecular basis underlying foxo1-mediated induction of il-1 in this study .
raw264.7 cells were mock - treated with pbs , or transduced with adv - foxo1 or control adv - null vector at a fixed dose ( moi , 200 pfu / cell ) .
after 24-h incubation in the presence of 100 ng / ml lps , conditioned medium was harvested for profiling the production of cytokine il-1 ( a ) , il-2 ( b ) , il-4 ( c ) , il-5 ( d ) , il-6 ( e ) , il-10 ( f ) , il-12 ( g ) , ifn- ( h ) , tnf- ( i ) , and granulocyte monocyte colony - stimulating factor ( gm - csf ) ( j ) .
* p < 0.001 vs. controls . to investigate the potential role of foxo1 in macrophage production of il-1 in vivo , we treated balb / c mice with once - daily intraperitoneal administration of lps ( 4 mg / kg ) for 2 days .
compared to pbs - treated control mice ( n = 8) , lps - treated mice ( n = 8) exhibited significantly elevated plasma il-1 levels ( fig .
two days after lps treatment , mice were killed and peritoneal macrophages were retrieved for real - time qrt - pcr analysis .
2b ) , correlating with significantly elevated il-1 mrna levels in peritoneal macrophages ( fig .
male balb / c mice ( 8 weeks old , n = 8 per group ) were treated with once - daily intraperitoneal injection of lps ( 4 mg / kg body weight ) or mock - treated with pbs for 2 days .
aliquots of blood ( 50 l ) were sampled from the tail vein for the determination of plasma il-1 levels ( a ) .
two days after lps treatment , peritoneal macrophages were isolated from individual mice for the preparation of total rna , which was subjected to real - time qrt - pcr assay for the determination of foxo1 ( b ) and il-1 ( c ) mrna levels using 18s rrna as control . * p <
0.05 and * * p < 0.001 vs. control . to determine whether foxo1 plays a role in linking insulin resistance to aberrant il-1 expression
, we studied foxo1 expression in peritoneal macrophages in obese db / db mice . because of the lack of leptin receptor , db / db mice are genetically obese , developing insulin resistance and hyperinsulinemia at weaning .
two groups of male db / db mice ( n = 7 , male , 10 weeks old ) and age- or sex - matched heterozygous db/+ lean littermates ( n = 7 ) were fasted for 16 h , as described ( 30 ) .
peritoneal macrophages were isolated from individual mice and subjected to real - time qrt - pcr analysis . as shown in fig .
3a , foxo1 expression was significantly upregulated in peritoneal macrophages of insulin - resistant obese db / db mice .
furthermore , db / db mice were associated with significantly higher levels of il-1 mrna in peritoneal macrophages ( fig .
3c ) , which was indicative of chronic inflammation in insulin - resistant obese db / db mice .
covariation of foxo1 and il-1 expression in peritoneal macrophages of insulin - resistant db / db mice .
peritoneal macrophages were isolated from insulin - resistant obese db / db mice ( n = 7 ) and control db/+ littermates ( n = 7 ) .
total macrophage rna was prepared and subjected to real - time qrt - pcr assay for the determination of foxo1 ( a ) and il-1 ( b ) mrna levels .
we hypothesized that the il-1 gene is a foxo1 target , and foxo1 promotes il-1 expression in macrophages in response to inflammation . to test this hypothesis , we treated raw264.7 cells with palmitate , the predominant saturated form of free fatty acid that is known to elicit low - grade inflammation .
4 , this treatment resulted in significant induction of il-1 production , as reflected in markedly elevated levels of il-1 protein in conditioned medium and il-1 mrna in cells .
/ l palmitate or bsa as control for 24 h. cells were harvested and subjected to real - time qrt - pcr assay for the determination of foxo1 ( a ) and il-1 ( b ) mrna levels .
a 2-kb dna fragment harboring the mouse il-1 promoter was cloned into the pgl3-basic luciferase reporter system ( fig .
il-1 promoter activity was determined in response to adenovirus - mediated foxo1 production in the presence and absence of insulin . as shown in fig .
5b , we detected a more than sixfold induction of the il-1 promoter activity in raw264.7 cells that were pretransduced with foxo1 vector .
this effect was counteracted by insulin , correlating with the ability of foxo1 to undergo insulin - dependent translocation from the nucleus to cytoplasm ( 25,27 ) .
a : a schematic depiction of the il-1 promoter  directed luciferase reporter system in phd386 .
raw264.7 cells in six - well microplates were transduced with adv - foxo1 or control adv - null vectors at a fixed dose ( moi , 200 pfu / cell ) , followed by transfection with 1-g phd386 plus 1-g pca35-lacz in the presence of 100 ng / ml lps in culture medium .
after 24-h incubation in the absence or presence of insulin ( 300 nmol / l ) , cells were harvested for the determination of luciferase and -galactosidase activities .
the relative luciferase activity was calculated by setting the ratio of luciferase/-galactosidase activity in control cells to 1 .
raw264.7 cells were transfected with wild - type ( wt ) or mutant ( mt ) il-1 promoter  directed luciferase system in the absence or presence of adenovirus - mediated foxo1 production .
after 24-h incubation in the presence of 100 ng / ml lps , cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls .
raw264.7 cells were cotransfected with phd386 and pca35-lacz in the absence or presence of adenovirus - mediated foxo1 production .
one subset of cells was transduced with 200 moi of adv - foxo1-rnai encoding foxo1-specific rnai or control adv - scrnai encoding scrambled rnai .
after 24-h incubation in the presence of 100 ng / ml lps , cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls .
the conditioned medium from the experiment in d was used for the determination of il-1 levels .
cells obtained from the experiment in d were subjected to semiquantitative immunoblot analysis for the determination of total foxo1 protein levels , using intracellular actin protein as control .
raw264.7 cells incubated in six - well dishes in the presence of lps ( 100 ng / ml ) for 24 h. cells were fixed with 1% formaldehyde , followed by chip assay using rabbit anti - foxo1 or control igg ( rabbit anti-galactosidase ) .
immunoprecipitates were analyzed by pcr assay for the detection of dna , using primers flanking the consensus foxo1 binding site within the il-1 promoter .
ns , not significant . to corroborate these findings , we altered the insulin - responsive element ( ire ) motif within the il-1 promoter via site - directed mutagenesis ( supplemental fig .
the resulting mutant il-1 promoter was analyzed for its ability to mediate the stimulatory effect of foxo1 on il-1 expression .
5c , alterations within the ire dna motif rendered the mutant il-1 promoter unresponsive to foxo1 production in raw264.7 cells .
as control , adenovirus - mediated foxo1 production resulted in a significant induction of wild - type il-1 promoter activity ( fig .
5c ) . to further prove that foxo1 contributed to il-1 regulation , we employed an rna interference ( rnai)-mediated gene silencing approach to knockdown foxo1 expression in cultured macrophages .
raw264.7 cells were transfected with phd386 expressing the il-1 promoter - directed luciferase reporter gene , followed by transduction of adenoviral vectors expressing foxo1-specific rnai or control scrambled rnai , as described ( 30 ) .
after 24-h incubation , cells were subjected to luciferase activity assay . as shown in fig .
5d , rnai - mediated foxo1 knockdown resulted in abolition of foxo1-mediated induction of il- promoter activity in lps - stimulated raw264.7 cells .
these results were corroborated when the conditioned medium was used for the determination of il-1 protein .
this effect was abolished by rnai - mediated foxo1 knockdown , as reflected in 80% of reduction in foxo1 protein levels in raw264.7 cells that were pretransduced with foxo1-rnai vector ( fig .
the above results spurred the idea that foxo1 stimulates il-1 expression via direct binding to the il-1 promoter .
5a ) , a consensus dna motif that is responsible for binding and mediating foxo1 induction of target gene expression ( 25,27 ) . interestingly , similar foxo1 consensus sites were detected in the regulatory region of both human and rat il-1 promoters , suggesting an evolutionally conserved mechanism ( supplemental fig .
s2 ) . to determine the ability of the conserved ire motif to mediate the stimulatory effect of foxo1 on il-1 promoter activity
, we performed chromatin immunoprecipitation ( chip ) assay to analyze the potential interaction of foxo1 with il-1 promoter dna in raw264.7 cells .
because of low foxo1 expression in unstimulated raw264.7 cells , we transduced raw264.7 cells with foxo1 vector in the presence of lps ( 100 ng / ml ) in culture medium .
after 24-h incubation , cells were subjected to chip assay using rabbit anti - foxo1 antibody or control rabbit igg .
the resulting immunoprecipitates were subjected to pcr analysis , using primers flanking the ire motif within the il-1 promoter .
a 457-bp dna corresponding to the ire region ( 1,359/902 nt ) of the il-1 promoter was produced from anti - foxo1 immunoprecipitates ( fig .
5f ) . in contrast , the immunoprecipitates derived from control igg were negative in the same pcr assay . to further illustrate the role of foxo1 in linking aberrant il-1 production to insulin resistance
, we determined the effect of lps or palmitate treatment on insulin signaling in cultured macrophages .
raw264.7 cells were incubated in the presence of lps ( 100 ng / ml ) or palmitate ( 0.2 mmol / l ) for 36 h , followed by the addition of insulin ( 300 nmol / l ) into culture medium .
the dose of insulin used in culture medium was based on our preliminary studies and data in the literature ( 36 ) .
cells were harvested 45 min after insulin action for the determination of insulin - stimulated phosphorylation of insulin receptor substrates ( irss ) and foxo1 , both of which are key signaling molecules downstream of insulin action . compared to mock - treated controls ,
raw264.7 cells pretreated with lps or palmitate treatment were associated with a significant reduction in insulin - stimulated phosphorylation of irs2 ( fig .
because of extremely low levels of irs1 expression in raw264.7 cells , we were unable to detect insulin - stimulated phosphorylation of irs1 .
( 37 ) , who show that irs1 is undetectable and irs2 is the sole substrate downstream of insulin action in primary murine macrophages .
lps or palmitate treatment also resulted in marked elevations of foxo1 production in raw264.7 cells ( fig .
however , the ratio of phosphorylated versus total foxo1 levels was significantly reduced , reflecting an impaired ability of insulin to promote foxo1 phosphorylation in lps- or palmitate - treated raw264.7 cells ( fig .
raw264.7 in six - well dishes were treated with either 100 ng / ml lps or 0.2 mmol / l palmitate .
aliquots of cell lyates were immunoprecipitated by anti - irs2 antibody , followed by immunoblot assay for the determination of phosphorylated irs2 using antiphosphotyrosine antibody ( a ) .
in addition , aliquots of cell lysates were subjected to immunoblot analysis for the determination of phosphorylated and total foxo1 in response to palmitate ( b ) or lps treatment ( c ) .
our findings of foxo1-stimulated il-1 production in inflammation - inflicted macrophages pose two potential mechanisms : 1 ) foxo1 exerts an independent effect on il-1 production , and 2 ) alternatively , foxo1 promotes il-1 overproduction through nf-b , a well - characterized pathway that is responsible for mediating proinflammatory cytokine production in macrophages under inflammatory or insulin - resistant conditions . to distinguish these two possibilities
, we chose to study the impact of foxo1 in nonstimulated macrophages . because of a concomitant induction of endogenous foxo1 and nf-b production in lps- or palmitate - treated macrophages , unstimulated raw264.7 cells were transduced with foxo1 or control vector to determine the net effect of foxo1 on il-1 expression in the absence of nf-b activation .
unexpectedly , foxo1 failed to stimulate il-1 production , as the amount of immunoreactive il-1 protein remained unchanged in foxo1 versus control vector  treated raw264.7 cells ( fig .
adenovirus - mediated foxo1 production resulted in significant induction of il-1 production in raw264.7 cells that were pre - exposed to lps ( fig .
to ascertain this finding , we transfected phd386 encoding the il-1 promoter - directed luciferase reporter system into nonstimulated raw264.7 cells .
no significant induction of luciferase activities in raw264.7 cells was detected in basal states ( fig .
7b ) , despite a threefold elevation in foxo1 protein levels in nave raw264.7 cells that were pretransduced with foxo1 vector ( fig .
after 24-h incubation in the absence of lps , the conditioned medium was collected for the determination of il-1 concentrations .
after 24-h incubation in the absence of lps , the relative luciferase activity in cells was determined using -galactosidase for the normalization of transfection efficiency .
cells from the experiment in b were subjected to immunoblot analysis for the determination of nuclear foxo1 protein levels .
directed luciferase reporter in the presence of foxo1 plus p50 or p65 production , using the dual luciferase system .
after 24-h incubation in the absence of lps , the relative promoter activity , defined as the ratio of firefly and renilla luciferase activities , was determined .
raw264.7 cells were incubated in the absence and presence of lps ( 100 ng / ml ) for 24 h , followed by immunoblot analysis using anti - foxo1 and anti - actin antibodies .
raw264.7 cells were transduced with foxo1 vector in the presence or absence of p50 production .
after 24-h incubation in the absence of lps , cells were fixed with 1% formaldehyde , followed by chip assay using rabbit anti - foxo1 antibody .
the immunoprecipitates were subjected to semiquantitative pcr assay using primers flanking the conserved ire dna motif within the il-1 promoter . after normalizing to input dna controls ,
raw264.7 cells were transfected with phd386 encoding the il-1 promoter  directed luciferase expression system in the presence or absence of foxo1 production , using the dual luciferase system .
one subset of cells was cotransfected with sirna that is targeted to the nf-b p50 subunit .
ns , not significant . to investigate whether foxo1 contributes to il-1 production via a mechanism that is dependent on nf-b , we determined the effect of foxo1 on il-1 promoter activity in the presence and absence of vector - mediated production of p50 or p65 , two subunits of nf-b . in keeping with our earlier observations ( fig .
7a ) , adenovirus - mediated foxo1 production exerted little effects on il-1 promoter activity in nonstimulated raw264.7 cells .
however , cotransfection of foxo1 along with p50 , but not with p65 , resulted in a significant induction of il-1 promoter activity ( fig .
these results suggest that foxo1 associates with p50 in stimulating il-1 expression in macrophages , or , alternatively , p50 is required for the recruitment of foxo1 to its target site to stimulate il-1 promoter activity . to address the first hypothesis
we show that lps , known to stimulate nf-b activation , also promoted foxo1 production in raw264.7 cells ( fig .
thus , to determine the net impact of foxo1 and p50 interaction on il-1 expression , we chose to perform the studies in unstimulated raw264.7 cells with vector - mediated production of foxo1 and p50 proteins .
foxo1-expressing raw264.7 cells were transfected with plasmid p50 encoding the human nf-b p50 subunit , followed by immunoprecipitation using anti - foxo1 or anti - p50 antibodies .
we detected the presence of foxo1 or p50 in immunoprecipitates that were derived from its cognate antibody , validating the method of immunoprecipitation .
however , neither foxo1 nor p50 was reciprocally coimmunoprecipitated by anti - p50 or anti - foxo1 antibodies ( data not shown ) .
these results argue against the notion that foxo1 formed a complex with p50 in promoting il-1 expression in macrophages .
we then addressed the second hypothesis that foxo1 binding to its target site within the il-1 promoter depends on p50 in cells .
implicit in this hypothesis is the presence of a highly conserved nf-b p50-binding site ( 5-gggagcatcc-3 ) downstream of the ire , the characteristic dna motif that is responsible for foxo1 binding ( fig .
we performed chip assay on raw264.7 cells in the presence of foxo1 or p50 expression alone or in combination .
the resulting immunoprecipitates were analyzed by semiquantitative pcr assay using specific primers flanking the foxo1 binding site in the il-1 promoter .
7f , positive interactions between foxo1 and il-1 promoter dna were detected in response to foxo1 or p50 production in raw264.7 cells .
however , coproduction of foxo1 and p50 resulted in a synergistic effect on foxo1 binding to its target site within the il-1 promoter .
this effect correlated with the costimulatory effect of foxo1 and p50 in combination on il-1 promoter activity ( fig .
to consolidate our findings that foxo1 signaling through p50 regulates il-1 production in macrophages , we used the sirna - mediated gene - silencing approach to knockdown p50 expression , followed by determining the ability of foxo1 to stimulate il-1 expression in lps - stimulated raw264.7 cells .
7 g , adenovirus - mediated foxo1 production resulted in a marked induction of luciferase activity , which was significantly reduced after sirna - mediated p50 knockdown in lps - stimulated raw264.7 cells .
these results suggest that depletion of nf-b p50 attenuated the ability of foxo1 to induce il-1 promoter activity in macrophages .
aberrant proinflammatory cytokine production is intertwined with insulin resistance in obesity and type 2 diabetes . to decipher the molecular basis that links proinflammatory cytokine production to insulin resistance , we determined the effect of foxo1 on cytokine production in macrophages , a cell type that plays a pivotal role in mediating whole - body insulin resistance in the face of low - grade inflammation .
macrophage raw264.7 cells were transduced with adv - foxo1 or adv - null vector at a predefined moi ( 200 pfu / cell ) , followed by the determination of cytokine concentrations in conditioned medium . as shown in fig .
1 , transduction of raw264.7 cells by foxo1 vector resulted in a significant induction of il-1 and il-2 production without alterations in the production of other cytokines including il-4 , il-5 , il-6 , il-12 , ifn- , tnf- , and granulocyte monocyte colony - stimulating factor .
in contrast , il-10 expression was significantly downregulated in response to foxo1 production in raw264.7 cells . in the absence of lps ,
the expression of proinflammatory cytokines remained at low basal levels independently of foxo1 action ( supplemental table s1 ) .
these data underscore the importance of foxo1 in regulating cytokine production in lps - activated macrophages . because of the close association between abnormal production of il-1 in macrophages and the pathogenesis of insulin resistance , we focused on the delineation of the molecular basis underlying foxo1-mediated induction of il-1 in this study .
raw264.7 cells were mock - treated with pbs , or transduced with adv - foxo1 or control adv - null vector at a fixed dose ( moi , 200 pfu / cell ) .
after 24-h incubation in the presence of 100 ng / ml lps , conditioned medium was harvested for profiling the production of cytokine il-1 ( a ) , il-2 ( b ) , il-4 ( c ) , il-5 ( d ) , il-6 ( e ) , il-10 ( f ) , il-12 ( g ) , ifn- ( h ) , tnf- ( i ) , and granulocyte monocyte colony - stimulating factor ( gm - csf ) ( j ) .
to investigate the potential role of foxo1 in macrophage production of il-1 in vivo , we treated balb / c mice with once - daily intraperitoneal administration of lps ( 4 mg / kg ) for 2 days .
compared to pbs - treated control mice ( n = 8) , lps - treated mice ( n = 8) exhibited significantly elevated plasma il-1 levels ( fig .
two days after lps treatment , mice were killed and peritoneal macrophages were retrieved for real - time qrt - pcr analysis .
2b ) , correlating with significantly elevated il-1 mrna levels in peritoneal macrophages ( fig .
male balb / c mice ( 8 weeks old , n = 8 per group ) were treated with once - daily intraperitoneal injection of lps ( 4 mg / kg body weight ) or mock - treated with pbs for 2 days .
aliquots of blood ( 50 l ) were sampled from the tail vein for the determination of plasma il-1 levels ( a ) .
two days after lps treatment , peritoneal macrophages were isolated from individual mice for the preparation of total rna , which was subjected to real - time qrt - pcr assay for the determination of foxo1 ( b ) and il-1 ( c ) mrna levels using 18s rrna as control .
to determine whether foxo1 plays a role in linking insulin resistance to aberrant il-1 expression , we studied foxo1 expression in peritoneal macrophages in obese db / db mice . because of the lack of leptin receptor , db / db mice are genetically obese , developing insulin resistance and hyperinsulinemia at weaning .
two groups of male db / db mice ( n = 7 , male , 10 weeks old ) and age- or sex - matched heterozygous db/+ lean littermates ( n = 7 ) were fasted for 16 h , as described ( 30 ) .
peritoneal macrophages were isolated from individual mice and subjected to real - time qrt - pcr analysis . as shown in fig .
3a , foxo1 expression was significantly upregulated in peritoneal macrophages of insulin - resistant obese db / db mice .
furthermore , db / db mice were associated with significantly higher levels of il-1 mrna in peritoneal macrophages ( fig .
3c ) , which was indicative of chronic inflammation in insulin - resistant obese db / db mice .
covariation of foxo1 and il-1 expression in peritoneal macrophages of insulin - resistant db / db mice .
peritoneal macrophages were isolated from insulin - resistant obese db / db mice ( n = 7 ) and control db/+ littermates ( n = 7 ) .
total macrophage rna was prepared and subjected to real - time qrt - pcr assay for the determination of foxo1 ( a ) and il-1 ( b ) mrna levels .
we hypothesized that the il-1 gene is a foxo1 target , and foxo1 promotes il-1 expression in macrophages in response to inflammation . to test this hypothesis , we treated raw264.7 cells with palmitate , the predominant saturated form of free fatty acid that is known to elicit low - grade inflammation .
4 , this treatment resulted in significant induction of il-1 production , as reflected in markedly elevated levels of il-1 protein in conditioned medium and il-1 mrna in cells .
raw264.7 cells were incubated in culture medium in the presence of 0.2 mmol / l palmitate or bsa as control for 24 h. cells were harvested and subjected to real - time qrt - pcr assay for the determination of foxo1 ( a ) and il-1 ( b ) mrna levels .
to characterize the underlying mechanism , we addressed whether foxo1 targets the il-1 promoter for trans - activation .
a 2-kb dna fragment harboring the mouse il-1 promoter was cloned into the pgl3-basic luciferase reporter system ( fig .
the resulting plasmid phd386 encoding the il-1 promoter  directed luciferase was transfected via electroporation into raw264.7 cells .
il-1 promoter activity was determined in response to adenovirus - mediated foxo1 production in the presence and absence of insulin . as shown in fig .
5b , we detected a more than sixfold induction of the il-1 promoter activity in raw264.7 cells that were pretransduced with foxo1 vector .
this effect was counteracted by insulin , correlating with the ability of foxo1 to undergo insulin - dependent translocation from the nucleus to cytoplasm ( 25,27 ) .
raw264.7 cells in six - well microplates were transduced with adv - foxo1 or control adv - null vectors at a fixed dose ( moi , 200 pfu / cell ) , followed by transfection with 1-g phd386 plus 1-g pca35-lacz in the presence of 100 ng / ml lps in culture medium .
after 24-h incubation in the absence or presence of insulin ( 300 nmol / l ) , cells were harvested for the determination of luciferase and -galactosidase activities .
the relative luciferase activity was calculated by setting the ratio of luciferase/-galactosidase activity in control cells to 1 .
raw264.7 cells were transfected with wild - type ( wt ) or mutant ( mt ) il-1 promoter  directed luciferase system in the absence or presence of adenovirus - mediated foxo1 production .
after 24-h incubation in the presence of 100 ng / ml lps , cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls .
raw264.7 cells were cotransfected with phd386 and pca35-lacz in the absence or presence of adenovirus - mediated foxo1 production .
one subset of cells was transduced with 200 moi of adv - foxo1-rnai encoding foxo1-specific rnai or control adv - scrnai encoding scrambled rnai .
after 24-h incubation in the presence of 100 ng / ml lps , cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls .
the conditioned medium from the experiment in d was used for the determination of il-1 levels .
cells obtained from the experiment in d were subjected to semiquantitative immunoblot analysis for the determination of total foxo1 protein levels , using intracellular actin protein as control .
raw264.7 cells incubated in six - well dishes in the presence of lps ( 100 ng / ml ) for 24 h. cells were fixed with 1% formaldehyde , followed by chip assay using rabbit anti - foxo1 or control igg ( rabbit anti-galactosidase ) .
immunoprecipitates were analyzed by pcr assay for the detection of dna , using primers flanking the consensus foxo1 binding site within the il-1 promoter .
, we altered the insulin - responsive element ( ire ) motif within the il-1 promoter via site - directed mutagenesis ( supplemental fig .
the resulting mutant il-1 promoter was analyzed for its ability to mediate the stimulatory effect of foxo1 on il-1 expression . as shown in fig .
5c , alterations within the ire dna motif rendered the mutant il-1 promoter unresponsive to foxo1 production in raw264.7 cells .
as control , adenovirus - mediated foxo1 production resulted in a significant induction of wild - type il-1 promoter activity ( fig .
to further prove that foxo1 contributed to il-1 regulation , we employed an rna interference ( rnai)-mediated gene silencing approach to knockdown foxo1 expression in cultured macrophages .
raw264.7 cells were transfected with phd386 expressing the il-1 promoter - directed luciferase reporter gene , followed by transduction of adenoviral vectors expressing foxo1-specific rnai or control scrambled rnai , as described ( 30 ) .
after 24-h incubation , cells were subjected to luciferase activity assay . as shown in fig .
5d , rnai - mediated foxo1 knockdown resulted in abolition of foxo1-mediated induction of il- promoter activity in lps - stimulated raw264.7 cells .
these results were corroborated when the conditioned medium was used for the determination of il-1 protein .
this effect was abolished by rnai - mediated foxo1 knockdown , as reflected in 80% of reduction in foxo1 protein levels in raw264.7 cells that were pretransduced with foxo1-rnai vector ( fig .
the above results spurred the idea that foxo1 stimulates il-1 expression via direct binding to the il-1 promoter .
5a ) , a consensus dna motif that is responsible for binding and mediating foxo1 induction of target gene expression ( 25,27 ) . interestingly , similar foxo1 consensus sites were detected in the regulatory region of both human and rat il-1 promoters , suggesting an evolutionally conserved mechanism ( supplemental fig .
s2 ) . to determine the ability of the conserved ire motif to mediate the stimulatory effect of foxo1 on il-1 promoter activity
, we performed chromatin immunoprecipitation ( chip ) assay to analyze the potential interaction of foxo1 with il-1 promoter dna in raw264.7 cells .
because of low foxo1 expression in unstimulated raw264.7 cells , we transduced raw264.7 cells with foxo1 vector in the presence of lps ( 100 ng / ml ) in culture medium .
after 24-h incubation , cells were subjected to chip assay using rabbit anti - foxo1 antibody or control rabbit igg .
the resulting immunoprecipitates were subjected to pcr analysis , using primers flanking the ire motif within the il-1 promoter .
a 457-bp dna corresponding to the ire region ( 1,359/902 nt ) of the il-1 promoter was produced from anti - foxo1 immunoprecipitates ( fig .
in contrast , the immunoprecipitates derived from control igg were negative in the same pcr assay .
to further illustrate the role of foxo1 in linking aberrant il-1 production to insulin resistance , we determined the effect of lps or palmitate treatment on insulin signaling in cultured macrophages .
raw264.7 cells were incubated in the presence of lps ( 100 ng / ml ) or palmitate ( 0.2 mmol / l ) for 36 h , followed by the addition of insulin ( 300 nmol / l ) into culture medium .
the dose of insulin used in culture medium was based on our preliminary studies and data in the literature ( 36 ) .
cells were harvested 45 min after insulin action for the determination of insulin - stimulated phosphorylation of insulin receptor substrates ( irss ) and foxo1 , both of which are key signaling molecules downstream of insulin action . compared to mock - treated controls ,
raw264.7 cells pretreated with lps or palmitate treatment were associated with a significant reduction in insulin - stimulated phosphorylation of irs2 ( fig .
because of extremely low levels of irs1 expression in raw264.7 cells , we were unable to detect insulin - stimulated phosphorylation of irs1 .
( 37 ) , who show that irs1 is undetectable and irs2 is the sole substrate downstream of insulin action in primary murine macrophages
. lps or palmitate treatment also resulted in marked elevations of foxo1 production in raw264.7 cells ( fig .
however , the ratio of phosphorylated versus total foxo1 levels was significantly reduced , reflecting an impaired ability of insulin to promote foxo1 phosphorylation in lps- or palmitate - treated raw264.7 cells ( fig .
raw264.7 in six - well dishes were treated with either 100 ng / ml lps or 0.2 mmol / l palmitate .
aliquots of cell lyates were immunoprecipitated by anti - irs2 antibody , followed by immunoblot assay for the determination of phosphorylated irs2 using antiphosphotyrosine antibody ( a ) .
in addition , aliquots of cell lysates were subjected to immunoblot analysis for the determination of phosphorylated and total foxo1 in response to palmitate ( b ) or lps treatment ( c ) .
our findings of foxo1-stimulated il-1 production in inflammation - inflicted macrophages pose two potential mechanisms : 1 ) foxo1 exerts an independent effect on il-1 production , and 2 ) alternatively , foxo1 promotes il-1 overproduction through nf-b , a well - characterized pathway that is responsible for mediating proinflammatory cytokine production in macrophages under inflammatory or insulin - resistant conditions . to distinguish these two possibilities
, we chose to study the impact of foxo1 in nonstimulated macrophages . because of a concomitant induction of endogenous foxo1 and nf-b production in lps- or palmitate - treated macrophages , unstimulated raw264.7 cells were transduced with foxo1 or control vector to determine the net effect of foxo1 on il-1 expression in the absence of nf-b activation .
unexpectedly , foxo1 failed to stimulate il-1 production , as the amount of immunoreactive il-1 protein remained unchanged in foxo1 versus control vector  treated raw264.7 cells ( fig .
, adenovirus - mediated foxo1 production resulted in significant induction of il-1 production in raw264.7 cells that were pre - exposed to lps ( fig .
1a ) . to ascertain this finding , we transfected phd386 encoding the il-1 promoter - directed luciferase reporter system into nonstimulated raw264.7 cells .
no significant induction of luciferase activities in raw264.7 cells was detected in basal states ( fig .
7b ) , despite a threefold elevation in foxo1 protein levels in nave raw264.7 cells that were pretransduced with foxo1 vector ( fig .
after 24-h incubation in the absence of lps , the conditioned medium was collected for the determination of il-1 concentrations .
b : raw264.7 cells were transfected with phd386 encoding the il-1 promoter  directed luciferase expression system in the presence or absence of foxo1 production .
after 24-h incubation in the absence of lps , the relative luciferase activity in cells was determined using -galactosidase for the normalization of transfection efficiency .
cells from the experiment in b were subjected to immunoblot analysis for the determination of nuclear foxo1 protein levels .
d : raw264.7 cells were transfected with phd386 encoding the il-1 promoter  directed luciferase reporter in the presence of foxo1 plus p50 or p65 production , using the dual luciferase system .
after 24-h incubation in the absence of lps , the relative promoter activity , defined as the ratio of firefly and renilla luciferase activities , was determined .
raw264.7 cells were incubated in the absence and presence of lps ( 100 ng / ml ) for 24 h , followed by immunoblot analysis using anti - foxo1 and anti - actin antibodies .
raw264.7 cells were transduced with foxo1 vector in the presence or absence of p50 production .
after 24-h incubation in the absence of lps , cells were fixed with 1% formaldehyde , followed by chip assay using rabbit anti - foxo1 antibody .
the immunoprecipitates were subjected to semiquantitative pcr assay using primers flanking the conserved ire dna motif within the il-1 promoter .
after normalizing to input dna controls , the amount of immunoprecipitated il-1 promoter dna fragments was compared among different conditions .
raw264.7 cells were transfected with phd386 encoding the il-1 promoter  directed luciferase expression system in the presence or absence of foxo1 production , using the dual luciferase system .
one subset of cells was cotransfected with sirna that is targeted to the nf-b p50 subunit .
to investigate whether foxo1 contributes to il-1 production via a mechanism that is dependent on nf-b , we determined the effect of foxo1 on il-1 promoter activity in the presence and absence of vector - mediated production of p50 or p65 , two subunits of nf-b . in keeping with our earlier observations ( fig .
7a ) , adenovirus - mediated foxo1 production exerted little effects on il-1 promoter activity in nonstimulated raw264.7 cells .
however , cotransfection of foxo1 along with p50 , but not with p65 , resulted in a significant induction of il-1 promoter activity ( fig .
these results suggest that foxo1 associates with p50 in stimulating il-1 expression in macrophages , or , alternatively , p50 is required for the recruitment of foxo1 to its target site to stimulate il-1 promoter activity . to address the first hypothesis
we show that lps , known to stimulate nf-b activation , also promoted foxo1 production in raw264.7 cells ( fig .
thus , to determine the net impact of foxo1 and p50 interaction on il-1 expression , we chose to perform the studies in unstimulated raw264.7 cells with vector - mediated production of foxo1 and p50 proteins .
foxo1-expressing raw264.7 cells were transfected with plasmid p50 encoding the human nf-b p50 subunit , followed by immunoprecipitation using anti - foxo1 or anti - p50 antibodies .
we detected the presence of foxo1 or p50 in immunoprecipitates that were derived from its cognate antibody , validating the method of immunoprecipitation .
however , neither foxo1 nor p50 was reciprocally coimmunoprecipitated by anti - p50 or anti - foxo1 antibodies ( data not shown ) .
these results argue against the notion that foxo1 formed a complex with p50 in promoting il-1 expression in macrophages .
we then addressed the second hypothesis that foxo1 binding to its target site within the il-1 promoter depends on p50 in cells .
implicit in this hypothesis is the presence of a highly conserved nf-b p50-binding site ( 5-gggagcatcc-3 ) downstream of the ire , the characteristic dna motif that is responsible for foxo1 binding ( fig .
we performed chip assay on raw264.7 cells in the presence of foxo1 or p50 expression alone or in combination .
the resulting immunoprecipitates were analyzed by semiquantitative pcr assay using specific primers flanking the foxo1 binding site in the il-1 promoter .
7f , positive interactions between foxo1 and il-1 promoter dna were detected in response to foxo1 or p50 production in raw264.7 cells . however , coproduction of foxo1 and p50 resulted in a synergistic effect on foxo1 binding to its target site within the il-1 promoter .
this effect correlated with the costimulatory effect of foxo1 and p50 in combination on il-1 promoter activity ( fig .
to consolidate our findings that foxo1 signaling through p50 regulates il-1 production in macrophages , we used the sirna - mediated gene - silencing approach to knockdown p50 expression , followed by determining the ability of foxo1 to stimulate il-1 expression in lps - stimulated raw264.7 cells .
7 g , adenovirus - mediated foxo1 production resulted in a marked induction of luciferase activity , which was significantly reduced after sirna - mediated p50 knockdown in lps - stimulated raw264.7 cells .
these results suggest that depletion of nf-b p50 attenuated the ability of foxo1 to induce il-1 promoter activity in macrophages .
our goal is to understand the molecular basis that links insulin resistance to abnormal production of proinflammatory cytokine il-1 in macrophages .
this effect was inhibited by insulin , which is correlated with the ability of foxo1 to undergo insulin - dependent phosphorylation and nuclear exclusion .
foxo1 was shown to bind to the il-1 promoter and enhance il-1 promoter activity . in accordance with these observations , there is a highly conserved foxo1 binding site within the il-1 promoter of humans , rats , and mice .
mutations of the foxo1 consensus site abrogated foxo1-mediated induction of il-1 promoter activity . in lps- or palmitate - treated macrophages , foxo1 expression along with its nuclear localization was increased , contributing to augmented foxo1 activity and elevated il-1 production .
lps or palmitate treatment also impaired the ability of insulin to promote irs2 and foxo1 phosphorylation in cultured macrophages .
these data characterize foxo1 at the interface between abnormal production of proinflammatory cytokine il-1 and the pathogenesis of insulin resistance in macrophages .
macrophage production of il-1 is governed by nf-b , a master regulator that integrates inflammatory signals to cytokine gene expression ( 11,38 ) . in unstimulated cells ,
nf-b is bound by ib and is sequestered in the cytoplasm . in the presence of inflammatory stimuli
, ib is phosphorylated by ib kinase and is destined for proteasome - mediated degradation . as a result
, nf-b is dissociated from ib and is translocated to the nucleus for promoting target gene expression ( 38 ) .
consistent with this notion is the conservation of a consensus nf-b target site within the il-1 promoter among different species ( supplemental fig .
s2 ) . however , this mechanism falls short of explaining why insulin resistance provokes the induction of il-1 production in activated macrophages . to address this fundamental issue ,
several independent studies show that activation of the pi3 kinase - akt pathway is effective in limiting lps - stimulated inflammatory cytokine production with uncharacterized mechanisms ( 3941 ) . here
we show that macrophage il-1 expression is regulated by foxo1 , a key nuclear transcriptional factor that mediates the inhibitory effect of insulin on target gene expression . in the absence of insulin , foxo1 acts in the nucleus as an enhancer for promoting target gene expression , whereas in the presence of insulin , foxo1 is phosphorylated by akt / pkb , resulting in its nuclear exclusion and contributing to the inhibition of target gene expression .
this phosphorylation - dependent protein trafficking mechanism is critical for insulin to regulate foxo1 transcriptional activity in cells ( 2527,30 ) .
our present studies shed light on the molecular basis that couples insulin resistance to the induction of proinflammatory cytokines .
it follows that in insulin - resistant states , loss of insulin inhibition of foxo1 activity consequently results in unrestrained il-1 expression in activated macrophages ( fig .
consistent with this interpretation , we show that foxo1 activity was significantly increased , correlating with impaired insulin action and elevated il-1 production in lps- and palmitate - stimulated macrophages .
furthermore , addition of insulin resulted in only partial suppression of il-1 promoter activity , which is indicative of insulin resistance in lps - stimulated raw264.7 cells ( fig .
, primary peritoneal macrophages retrieved from lps - treated mice and insulin - resistant db / db mice were invariably associated with increased foxo1 activity , accompanied by elevated il-1 levels in plasma .
tripathy et al . ( 42 ) show that acute elevation of free fatty acid directly provokes inflammation , culminating in enhanced nf-b activity and increased proinflammatory cytokine expression in mononuclear cells in healthy individuals . these findings together with our data
support the notion that circulating mononuclear cells in insulinresistant obese subjects are in a proinflammatory state ( 43 ) . indeed , treatment with thiazolidenediones
, a class of insulin sensitizers for improving peripheral insulin resistance , reduces mononuclear nf-b activity and ameliorates inflammation in obese subjects ( 4446 ) .
foxo1 targets at the ire dna motif of the il-1 promoter for trans - activation . under
insulin - resistant or inflammatory conditions , foxo1 activity is increased because of impaired abilities of insulin to phosphorylate and translocate foxo1 from the nucleus to cytoplasm .
this effect along with the activation of the nf-b pathway synergistically promotes macrophage production of proinflammatory cytokine il-1. the evolutionally conserved ire dna motif and nf-b binding site are indicated within the promoter - proximal region .
although foxo1 targeted il-1 promoter for trans - activation , foxo1 did not elicit a significant induction of il-1 expression in cultured raw264.7 cells in the absence of inflammatory stimuli . in response to inflammatory signals such as lps or palmitate
this effect was mirrored by the activation of nf-b in lps- or palmitate - activated macrophages .
interestingly , we show that foxo1 in concert with activated nf-b produced a synergistic effect on the induction of il-1 overproduction in the presence of inflammation . together
these results suggest that augmented foxo1 activity serves to amplify the action of nf-b in stimulating macrophage il-1 production under inflammatory conditions ( fig .
first , the productive interaction between foxo1 and il-1 promoter dna was significantly enhanced in the presence of p50 production , correlating with the costimulatory effect of foxo1 and p50 on il-1 promoter activity in cultured macrophages .
second , sirna - mediated knockdown of p50 significantly attenuated the ability of foxo1 to stimulate il-1 expression in lps - stimulated macrophages .
third , the il-1 promoter contains both foxo1 and nf-b target sites that are juxtaposed within the promoter - proximal region of the il1b gene in mice , rats , and humans ( supplemental fig .
s2 ) , implying an evolutionally conserved mechanism for foxo1 signaling through the nf-b pathway in regulating proinflammatory cytokine il-1 production in macrophages in response to insulin resistance . in conclusion
, we characterized the il1b gene as a foxo1 target , demonstrating that macrophage production of il-1 was stimulated by foxo1 and inhibited by insulin . under insulin - resistant or inflammatory conditions ,
foxo1 activity was augmented , because of an impaired ability of insulin to phosphorylate foxo1 and promote its nuclear exclusion .
this effect along with active nf-b contributed to macrophage overproduction of il-1. although insulin resistance is associated with low - grade inflammation in obesity and type 2 diabetes , the underlying mechanism remains obscure .
our data suggest that foxo1 signaling through nf-b plays a significant role in coupling insulin resistance to proinflammatory cytokine il-1 production in obesity and type 2 diabetes .
 objectivemacrophages play an important role in the pathogenesis of insulin resistance via the production of proinflammatory cytokines . 
 our goal is to decipher the molecular linkage between proinflammatory cytokine production and insulin resistance in macrophages.research design and methodswe determined cytokine profiles in cultured macrophages and identified interleukin ( il)-1 gene as a potential target of foxo1 , a key transcription factor that mediates insulin action on gene expression . 
 we studied the mechanism by which foxo1 mediates insulin - dependent regulation of il-1 expression in cultured macrophages and correlated foxo1 activity in peritoneal macrophages with il-1 production profiles in mice with low - grade inflammation or insulin resistance.resultsfoxo1 selectively promoted il-1 production in cultured macrophages . 
 this effect correlated with the ability of foxo1 to bind and enhance il-1 promoter activity . 
 mutations of the foxo1 binding site within the il-1 promoter abolished foxo1 induction of il-1 expression . 
 macrophages from insulin - resistant obese db / db mice or lipopolysaccharide - inflicted mice were associated with increased foxo1 production , correlating with elevated levels of il-1 mrna in macrophages and il-1 protein in plasma . in nonstimulated macrophages , foxo1 remained inert with benign effects on il-1 expression . in response to inflammatory stimuli , 
 foxo1 activity was augmented because of an impaired ability of insulin to phosphorylate foxo1 and promote its nuclear exclusion . 
 this effect along with nuclear factor-b acted to stimulate il-1 production in activated macrophages.conclusionsfoxo1 signaling through nuclear factor-b plays an important role in coupling proinflammatory cytokine production to insulin resistance in obesity and diabetes . 