metabolic syndrome describes a combination of metabolic abnormalities that include central obesity , dyslipidemia , hypertension , insulin resistance , and a pro - inflammatory and pro - thrombotic state .
an important group of pharmacological targets for the treatment of metabolic syndrome are the peroxisome proliferator activated receptors ( ppars ) .
ppars are ligand - activated transcription factors belonging to the superfamily of nuclear receptors , which include numerous cellular receptors for nutrients and steroids .
so far , three ppar isotypes (  , / ,  ) have been identified in a wide range of species , each displaying a different tissue distribution and ligand specificity .
ppars share a similar structure and a common molecular mechanism of action by forming an obligate heterodimer with the 9-cis retinoic acid receptor rxr .
ppar - rxr heterodimers selectively bind genomic sequences consisting of a direct repeat of the hexameric nucleotide sequence aggtca separated by 1 nucleotide ( direct repeat-1 ) .
these so - called peroxisome proliferator response elements ( ppre ) are located in the promoter of ppar target genes or in intronic regions [ 25 ] .
the ppar isotype ( nr1c1 ) is highly expressed in liver , and governs the adaptive response to fasting [ 68 ] .
ppar is an extremely important regulator of hepatic nutrient metabolism including fatty acid oxidation ( peroxisomal and mitochondrial ) , fatty acid uptake , amino acid metabolism , glycerol metabolism , and lipoprotein assembly and transport [ 911 ] .
in addition , ppar potently suppresses the hepatic inflammatory response [ 12 , 13 ] , an effect that is also observed in extra - hepatic tissues such as the vascular wall .
much less is known about the role of ppar in other tissues , although evidence is accumulating that ppar induces cardiac and skeletal muscle fatty acid oxidation [ 15 , 16 ] .
importantly , ppar mediates the effects of hypolipidemic fibrate drugs , which decrease plasma triglycerides and increase plasma hdl concentrations .
in contrast to ppar , ppar ( nr1c3 ) is highly expressed in white adipose tissue ( wat ) , where it promotes lipid storage .
ppar is a key transcription factor in the adipogenesis program and is essential for adipocyte survival [ 17 , 18 ] .
it also serves as the molecular target for the thiazolidine - dione ( tzd ) class of insulin - sensitizing drugs that are widely used in the treatment of type 2 diabetes .
ppar promotes whole body glucose utilization ; however , it has been difficult to identify the molecular mechanisms behind this effect .
much of the attention has been focused on possible cross - talk between adipose tissue and skeletal muscle , as muscle is responsible for the major share of whole body glucose utilization .
however , adipose tissue is a large organ , especially in the obese , and accordingly it can also be envisioned that the insulin - sensitizing effect of tzds on glucose uptake is partially exerted at the adipose tissue level .
while ppar and ppar have been extensively studied over many years , much less is known about the function of the ppar/ isotype ( nr1c2 ) .
studies with genetically modified ppar/ mice have illustrated the importance of this nuclear receptor in wat and skeletal muscle , two organs that have a key role in glucose homeostasis [ 1922 ] .
it was shown that activation of ppar/ in adipose tissue protects against adiposity and hyperlipidemia by inducing fatty acid catabolism . moreover , pharmacological activation as well as specific constitutive over - expression of ppar/ leads to a shift in muscle fiber composition towards type i muscle fibers , resulting in increased muscle oxidative capacity [ 20 , 22 ] .
ppar/ has also been shown to stimulate hepatic vldl production , influence wound healing , and affect colon carcinogenesis [ 2325 ] .
however , whether ppar/ has a functional role in glucose homeostasis , in analogy with other ppar isotypes , remains to be firmly established .
here we show that glycogen synthase 2 ( gys-2 ) , the rate - limiting enzyme for glycogen synthesis in liver and adipose tissue , is a target gene of ppar , ppar/ and ppar. transcriptional regulation is achieved via a ppar response element present in the first intron .
we show that an additional direct repeat response element identified in the gys-2 promoter mediates transactivation by hepatic nuclear factor 4 ( hnf4 ) .
chemicals . wy14643 was obtained from eagle picher technologies laboratories ( lenexa , ks , usa ) .
dulbecco s modified eagles medium ( dmem ) , fetal calf serum ( fcs ) , calf serum and penicillin / streptomycin / fungizone were from cambrex bioscience ( seraing ) .
otherwise , chemicals were from sigma ( zwijndrecht , the netherlands ) . animal experiments .
ppar/ mutant null mice ( ppar/-/- ) and ppar heterozygous mice ( ppar+/ ) were on a mixed background ( svl29/c57bl/6 ) and have been described previously [ 26 , 27 ] .
ppar-/- mice and corresponding wild - type mice on an svl29 background were purchased at jackson laboratories ( bar harbor , me , usa ) .
liver - specific hepatocyte nuclear factor 4 ( hnf4)-null mice were generated as described previously .
livers were collected from 45-day - old hnf4 x albumin - cre ( ko ) and hnf4 x albumin - cre ( flox ) mice .
for the fasting experiment , 3-month - old male mice were fasted for different periods of time starting at the onset of the light cycle . for the refeeding experiment ,
mice were fasted for 24 h after which they were put back on chow for 7 h before sacrifice . after sacrificing the animals ,
the animal experiments were approved by the animal experimentation committee of wageningen university or the etat de vaud ( switzerland ) .
total rna was prepared from epididymal wat of wild - type and ppar/-/- mice ( five animals of each genotype ) using trizol reagent ( invitrogen , breda , the netherlands ) and subsequently pooled per group .
pooled rna was further purified using qiagen rneasy columns , and the quality was verified using bioanalyzer 2100 ( agilent , amsterdam ) . for
one cycle crna synthesis ( affymetrix , santa clara , usa ) 10 g of rna was used .
hybridization , washing and scanning of affymetrix genechip mouse genome 430 2.0 arrays was according to standard affymetrix protocols .
fluorimetric data were processed by affymetrix genechip operating software and the gene chips were globally scaled to all the probe sets with an identical target intensity value .
3t3-l1 fibroblasts were grown in dmem plus 10 % calf serum and plated for final differentiation in dmem plus 10 % fcs . at 2 days after reaching confluence , the medium was changed and the following compounds were added : isobutyl methylxanthine ( 0.5 mm ) , dexamethasone ( 1 m ) , and insulin ( 5 g / ml ) . after 3 days , the medium was changed to dmem plus 10% fcs and insulin ( 5 g / ml ) . after 6 days the medium
briefly , after cannulation of the portal vein , the liver was perfused with calcium - free hbss , which was pre - gassed with 95 % o2 /5 % co2 .
next , the liver was perfused with a collagenase solution until swelling and degradation of the internal liver structure was observed .
cells were cultured in william s medium e supplemented with 10% fcs , penicillin / streptomycin / fungizone , insulin and dexamethasone .
cells were plated in collagen ( serva feinbiochemica , heidelberg , germany ) -coated wells with a density of 0.510 cells / ml . after 4 h of incubation , the medium was removed and replaced with fresh medium .
total rna was extracted from tissues with trizol reagent ( invitrogen ) ; 1 g total rna was then reverse - transcribed with iscript ( bio - rad , veenendaal , the netherlands ) .
cdna was pcr - amplified with platinum taq dna polymerase ( invitrogen ) on a bio - rad icycler or mylq pcr machine .
primers were designed to generate a pcr amplification product of 100200 bp and were taken from primerbank ( http://pga.mgh.harvard.edu/primerbank/ ) .
specificity of the amplification was verified by melt curve analysis and evaluation of efficiency of pcr amplification .
expression was related to the control gene 36b4 , which did not change under any of the experimental conditions studied .
the following primer pairs were used : mgys-2 ( forward ) : ccagcttgacaagttcgaca , mgys-2 ( reverse ) : at - caggcttcctcttcagca , m36b4 ( forward ) : agcgcgtcctggcattgtgtgg , m36b4 ( reverse ) : gggcagcagtggtggcagcagc , mppar ( forward ) : tattcggctgaagctggtgtac , mppar ( reverse ) : ctggcatttgttccggttct , mppar ( forward ) : ttgagcccaagttcgagtttg , mppar ( reverse ) : cggtctccacacagaatgatg , mppar ( forward ) : ca - caatgccatcaggtttgg , mppar ( reverse ) : gctggtcgatatcactggagatc .
the proximal part of the mouse gys-2 promoter was pcr amplified from mouse genomic dna ( strain c57bl/6 ) using the forward primer : 5 cttgctgcctttcag - gagagggcag 3 and reverse primer : 5 ttctctttagc - catta ag atag 3. the resulting 553-bp fragment was used for a second pcr amplification step introducing hindlll and kpnl sites , which were used for subcloning into the pgl-3 basic vector ( invitrogen ) .
a 156-bp nucleotide fragment surrounding the putative ppre within the mgys-2 promoter was pcr amplified from mouse genomic dna ( strain c57bl/6 ) and subcloned into kpnl / bg / ii sites of the pgl3 sv40 promoter vector ( pgl3-tk - luc , promega , leiden , the netherlands ) using the forward primer : 5aaatcg - cagctgaaacct 3 , and reverse primer : 5 ctcctgcttgtgcttctgc 3. a 314-nucleotide fragment surrounding the putative ppre within intron 1 of the mouse gys-2 gene was pcr amplified from mouse genomic dna ( strain c57bl/6 ) and subcloned into the kpnl and bglll sites of the pgl - tk - luc reporter gene .
reporter vectors were transfected into human hepatoma hepg2 cells , together with an expression vector ( psg5 ) for mppar , mppar , or mpparyl , in the presence or absence of wy14643 ( 50 m ) , l-165041 ( 5 m ) , or rosiglitazone ( 5 m ) , respectively .
a -galactosidase reporter vector was co - transfected to normalize for differences in transfection efficiency .
luciferase activity was measured 24 h post transfection using the promega luciferase assay kit ( promega ) on a fluoroskan ascent fl apparatus ( thermo labsystems , breda , the netherlands ) .
-galactosidase activity was measured in the cell lysate by a standard assay using 2-nitrophenyl-d - galactopyrano - side as a substrate . to disable the mouse gys-2 ppre within the mgys-2 promoter , two
separate ( a and b ) partially overlapping pcr fragments were generated using the wild - type mgys-2 promoter as a template .
primers sets used to generate part a of the mutated mgys-2 promoter fragment were : 5-tttggtctaaaggcctttggccaaagg-3 and 5-cttgctgcctttcaggagagggc ag-3. primers sets used to generate part b of the mutated mgys-2 promoter fragment were : 5-cctttggccaaaggccttta - gaccaaa-3 and 5-ttctctttagccattaagatagg - gattg-3. pcr was carried - out using the two upper dna fragments and the following primers : 5- cccaagcttcttgctgcctttcaggag-3 and 5- ggggtaccttctctttagccattaagatag-3. the pcr fragment was subsequently cloned into the pgl3 basic reporter vector ( hindiii / kpni cloning site ) and verified by automated sequencing .
the hhnf4 expression plasmid was constructed by amplifying human hepatoma hepg2 cdna using the following primers , forward primer 5-gaatgcgactctccaaaacc-3 and reverse primer 5-atccttcccattcctgctct-3 , followed by subcloning of the resulting pcr product into an pgem - teasy vector ( promega ) .
the insert was excised by noti digestion and further subcloned into pcdna3.1/v5-hisa ( invitrogen ) .
hrxr and mppar proteins were generated from psg5 expression vectors , using the tnt coupled in vitro transcription / translation system ( promega ) .
the following oligos were annealed to generate the double - stranded dna probe ; for dr - lint : 5-caggactttggtgacctctggcctatat-3 and 5-acacatataggccagaggtcaccaaagtc-3. for nonspecific competition , the following primers were used : etsf 5-tggaatgtaccggaaataacacca-3 , etsr 5- tggtgttatttccggtacattcca-3. oligonucleotides were annealed and labeled by klenow filling ( new england biolabs , leusden , the netherlands ) using redivue [ -p]dctp ( 3000 ci / mmol ; amersham , roosendaal , the netherlands ) . in vitro - translated proteins ( 0.50.8 l / reaction ) were pre - incubated for 15 min on ice in 1 binding buffer ( 80 mm kcl , 1 mm dtt , 10 mm tris - hcl ph 7.4 , 10 % glycerol , plus protease inhibitors ) in presence of 2 g poly[di.dc ] , 5 g sonicated salmon sperm dna and competitor oligonucleotides in a final volume of 20 l .
then 1 ng ( 1 ng/l ) of radiolabeled oligo was added and incubation was continued for another 10 min at room temperature .
complexes were separated on a 4 % polyacrylamide gel ( acrylamide / bisacrylamide 37.5:1 ) equilibrated in 0.5 tbe at 25 ma .
chromatin immunoprecipitation ( chip ) on 3t3-l1 cells and mouse liver was carried out as described previously .
sequences of primers used for pcr were 5-tctggcaggcataaggacccgagtt-3 and 5- ggaagccaggacagagtgcaaatacaat-3 for dr - lint ( intron ) . for dr - lprom ,
the following primers were used : 5-aaaactgcttgtgtctgagggaaac-3 and 5-agag - gacagactgagcatgacaagag-3. control primers used were 5-gctgcgagatccatcacccactaaac-3 and 5-agccatctcaccagccccaactt-3. antibodies against ppars were from santa cruz biotechnology ( santa cruz , ca , usa ) .
chip on rat hepatocytes was done using a commercially available kit ( active motif , rixensart , belgium ) .
the primers used to amplify the sequence surrounding the dr - lprom were 5-gaatgccgctgtgcctgagggaaac-3 and 5-agaggacagaagaagagtgacaagag-3. for dr - lint : 5-tctgtcaggcataaggacctgggtt-3 and 5-attgtatttgaactctgtcctggtctct-3. histology .
liver tissue from wild - type and ppar-/- mice was embedded in tissue - tek o.c.t compound from sakura finetek ( zoeterwoude , the netherlands ) and frozen .
cryosections of 5 m from frozen liver were made and analyzed for glycogen accumulation using the periodic acid - schiff ( pas ) reaction .
a mouse anti - glycogen synthase monoclonal antibody was used ( clone gs-7h5 mab3106 ) ( chemicon international , hampshire , uk ) .
the primary antibody was used at a dilution of 1:1000 and the secondary antibody ( anti - mouse igg , dako , glostrup , denmark ) was used at a dilution of 1:8000 .
accordingly , we compared gene expression in wat of wild - type versus ppar/-/- mice using affymetrix micro - array analysis .
the expression of several genes involved in glucose and lipid metabolism was down - regulated in ppar/-/-mice , including ppar , pgc - l , and glut4 , which was confirmed for several genes by real - time quantitative pcr ( qpcr ) ( table 1 ) .
expression of gys-2 was most significantly down - regulated in ppar/-/-mice , and therefore gys-2 was selected for more detailed investigation .
q - pcr confirmed the marked down - regulation of gys-2 mrna in wat of ppar/-/- mice ( fig .
furthermore , expression of gys-2 also appeared to be down - regulated in wat of ppar+/ and ppar-/- mice , although the former result did not achieve statistical significance . in ppar/-/- mice ,
the decrease in gys-2 mrna was paralleled by a significant down - regulation of ppar and ppar expression , while in ppar mice expression of ppar/ was significantly down - regulated ( fig .
these data show that ppars are crucial for maintaining gys-2 expression in fat , although it is difficult to ascertain which ppar isotype is the main regulator of gys-2 expression in wat . 

figure 1expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) .
expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr .
the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) .
( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr .
data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist.table 1genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice.geneproductfold decrease micro - arrayq - pcrglucose metabolismgys2glycogen synthase 213.279.28pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.99pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.64gys3glycogen synthase 3 , brain2.25ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen2.19slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.161.56slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.041.56pfk-26-phosphofructo-2-kinase / fructose-2,6-bisphosphatase2.01gdc1glycerol phosphate dehydrogenase 1 , cytoplasmic adult1.93ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen1.91gdc1glycerolphosphate dehydrogenase 1 , cytoplasmicadult1.84pyglliver glycogen phosphorylase1.71lipid metabolismppargperoxisome proliferator activator receptor gamma2.993.63c5dsterol - c5-desaturase2.85lrp1low density lipoprotein receptor - related protein 12.22cd36cd36 antigen2.203.09]fads3fatty acid desaturase 32.06lipelipase , hormone sensitive1.961.66lrp2low density lipoprotein receptor - related protein 21.91fabp5fatty acid binding protein 5 , epidermal1.87dgat1diacylglycerol acyltransferase1.87decr12,4-dienoyl coa reductase 1 , mitochondrial1.85slc27a1solute carrier family 27 ( fatty acid transporter ) , member 11.85phyhphytanol - coa hydroxylase1.77dgat2/1diacylglycerol o - acyltransferase 2-like 11.75ppargc1peroxisome proliferative activated receptor , gamma , coactivator 11.741.97 expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) .
expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr .
the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) .
( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr .
data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist .
genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice . to investigate whether expression of gys-2 in adipocytes is under direct control of ppars , the effect of ppar ligands on gys-2 mrna was studied in differentiated mouse 3t3-l1 adipocytes .
it was observed that the ppar/ agonist l165041 , and the ppar agonists ciglitazone and rosiglitazone significantly induced gys-2 mrna levels ( fig .
thus , gys-2 may represent a direct target gene of ppar and ppar/ in adipocytes .
it was observed that liver glycogen levels were reduced in refed ppar-/- mice compared to refed wild - type mice [ 30 , 31 ] , which we confirmed using histochemical staining ( fig .
expression of gys-2 is highest in liver , followed by wat ( our unpublished data ) .
we confirm that hepatic gys-2 mrna is markedly reduced in ppar-/- mice ; however , only in the 24-h fasted and refed state ( fig .
3a ) . to further examine the role of ppar in gys-2 expression , primary hepatocytes from wild - type and ppar-/- mice were treated with the synthetic ppar agonist wy14643 , allowing for a direct evaluation of the effect of ppar activation on gys-2 expression .
basal expression of gys-2 was about fourfold reduced in ppar-/- hepatocytes , indicating a requirement for ppar ( fig .
furthermore , wy14643 stimulated gys-2 expression in wild - type but not in ppar-/- hepatocytes ( fig .
3b ) . induction of gys-2 expression by synthetic ppar agonists was also observed in rat primary hepatocytes ( fig .
3c ) . together , these data suggest a direct role of ppar in governing hepatic gys-2 expression . 

figure 2staining for hepatic glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice .
mice were fasted for 24 h followed by refeeding for 7 h before sacrifice.figure 3ppar governs hepatic expression of gys-2 .
( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr .
mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting .
significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr .
significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) .
( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) .
the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) .
glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice .
( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr .
mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting .
significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr .
significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) .
( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) .
the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) .
identification of a putative ppre in the proximal promoter of the mouse gys-2 gene . to determine what genomic region could be responsible for the ppar - induced up - regulation of gys-2 mrna
, the mouse gys-2 gene was scanned for potential ppres ( nubiscan algorithm and hidden markov model framework ) [ 33 , 34 ] .
a direct repeat-1 motif ( dr - lprom ) was localized to the proximal gys-2 gene promoter , about 169 bp upstream from the transcription start site . with the exception of two nucleotides ,
dr - lprom is identical to the consensus sequence , suggesting that this sequence could serve as a functional ppre ( fig .
figure 4the dr-1 present in the gys-2 promoter does not mediate ppar - dependent transactivation .
( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint .
( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone .
chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) .
the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes .
( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint .
( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone .
chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) .
the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes .
( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . to examine whether the promoter region containing the putative ppre is responsible for ppar - dependent up - regulation of gys-2 expression , a 553-nucleotide fragment of the mouse gys-2 promoter gene
was cloned in front of a luciferase reporter gene and transactivation studies were carried out in hepg2 cells .
surprisingly , co - transfection of ppar or pparl expression vectors in combination with ppar agonists slightly decreased luciferase activity , while ppar/ activation had little effect ( fig .
transactivation assays performed with a small genomic fragment surrounding dr - lprom cloned in front of sv40-luciferase led to a similar overall ppar - mediated repression for ppar and pparl , while ppar/ had little effect ( fig .
co - transfection of rxr or of different co - activators such as cbp and pgcl did not change this pattern ( data not shown ) .
thus , the ppre identified in the gys-2 promoter probably does not mediate the effect of ppars on gys-2 expression .
nevertheless , chip experiments carried out in 3t3-l1 cells indicated that ( 1 ) ppar was bound to dr - lprom in mature adipocytes , but not in pre - adipocytes ( fig .
4d ) , and ( 2 ) ppar/ was bound to dr - lprom in pre - adipocytes and , more strongly , in mature adipocytes ( fig .
4e ) . thus , despite dr - lprom behaving poorly as a ppre in classical transactivation assay , it binds both ppar and ppar/ in adipocytes .
this suggests that in vivo binding of ppar and ppar/ to dr - lprom does not translate into transcriptional activation of the gys-2 gene , and accordingly that activation of gys-2 expression by ppars may be mediated by another genomic region .
it should be mentioned that chip did not reveal any binding of ppar to dr - lprom in hepatocytes ( data not shown ) .
interestingly , using the same strategy as described above , a putative ppre that is homologous to the consensus dr-1 sequence was identified in intron 1 of the mouse gys-2 gene ( fig .
4a ) . to assess whether dr - lint was able to mediate ppar - dependent transactivation , a 314-nucleotide genomic fragment surrounding dr - lint was cloned in front of the sv40 promoter followed by a luciferase reporter gene . in hepg2 cells treatment with the synthetic ppar agonist
wy14643 induced reporter activity and this activation was further enhanced upon co - transfection of mppar ( fig .
similar inductions of reporter activity were observed for ppar/ and ppar and their respective agonists ( fig .
thus , dr - lint is able to mediate ppar - dependent transactivation , irrespective of the ppar isotype , suggesting that it may at least be partially responsible for ppar - dependent regulation of gys-2 expression . 

figure 5gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) .
( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone .
( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing
gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein .
fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated .
gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) .
( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone .
( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing
gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein .
fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . in agreement with the transactivation data , ppar , ppar/ and ppar proteins
a retarded heterodimeric complex was observed only in the presence of both ppar and obligate binding partner rxr ( fig .
the complex disappeared in the presence of an excess of cold specific oligonucleotide , but not nonspecific oligonucleotide .
examination of in vivo ppar binding to dr - lint by chip yielded very similar results as for dr - lprom : ppar was bound to dr - lint in mature 3t3-l1 adipocytes , but not in pre - adipocytes ( fig .
6a ) , whereas ppar/ was bound to dr - lint in both pre- and mature adipocytes ( fig .
chip analysis demonstrated binding of ppar to dr - lint in wild - type but not ppar mice , and binding was enhanced by fasting and wy14643 ( fig .
together , these data indicate that mouse gys-2 is a direct ppar target gene and that regulation by ppars is at least partially mediated by a ppre present in intron 1 . 

figure 6ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo .
chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) .
the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) .
pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo .
chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) .
the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) .
pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
gys-2 is a novel direct target of the liver enriched factor hnf4. as explained above , ppar caused a reduction in gys-2 promoter activity via dr - lprom ( fig .
a similar decrease of promoter activity in response to ppar despite the presence of a putative ppre has been reported for other genes .
indeed , it was found that ppar decreases expression of the apociii and transferrin genes via competition with the hnf4. since hnf4 is known to recognize dr-1 sequences as well , we examined whether hnf4 might control the expression of gys-2 in liver , possibly via dr - lprom .
gys-2 mrna levels were markedly decreased in liver - specific hnf4null mice , thus supporting a role for hnf4 in regulating gys-2 expression ( fig .
7a ) . in transactivation assays using the gys-2 promoter , hnf4 markedly activated reporter activity , suggesting the presence of a hnf4 response element within the 0.55-kb promoter fragment ( fig .
7b ) . mutating dr - lprom resulted in an approximately 50 % reduction in hnf4-dependent activation of the gys-2 promoter ( fig .
7c ) , which suggests that ( a ) hnf4 responsiveness is partially mediated by dr - lprom , or ( b ) the mutations with dr - lprom only partially disabled hnf4 responsiveness .
regardless of these explanations , these data suggest that hnf4 directly regulates the hepatic expression of gys-2 at least partially via dr - lprom .
finally , chip clearly showed hnf4 binding to dr - lprom but not dr - lint in rat primary hepatocytes ( fig .
figure 7gys-2 dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) .
( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes .
( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector .
normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 .
dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) .
( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes .
( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector .
normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 .
whereas hnf4 activates the gys-2 promoter via dr - lprom , ppar does the opposite , suggesting that ppar may interfere with gys-2 promoter activation by hnf4. to examine whether this is the case , the effect of ppar on hnf4-mediated transactivation of the 0.55-kb gys-2 promoter was studied .
ppar activation significantly reduced hnf4-dependent transactivation , indicating competition between hnf4 and ppar in the regulation of the gys-2 promoter ( fig .
as already mentioned above , we failed to find any evidence for binding of ppar to dr - lprom in hepatocytes .
thus , the inhibitory effect of ppar on transcriptional activation of gys-2 by hnf4 likely does not occur via competition with hnf4 for actual binding to dr - lprom .
accordingly , we compared gene expression in wat of wild - type versus ppar/-/- mice using affymetrix micro - array analysis .
the expression of several genes involved in glucose and lipid metabolism was down - regulated in ppar/-/-mice , including ppar , pgc - l , and glut4 , which was confirmed for several genes by real - time quantitative pcr ( qpcr ) ( table 1 ) .
expression of gys-2 was most significantly down - regulated in ppar/-/-mice , and therefore gys-2 was selected for more detailed investigation .
q - pcr confirmed the marked down - regulation of gys-2 mrna in wat of ppar/-/- mice ( fig .
furthermore , expression of gys-2 also appeared to be down - regulated in wat of ppar+/ and ppar-/- mice , although the former result did not achieve statistical significance . in ppar/-/- mice ,
the decrease in gys-2 mrna was paralleled by a significant down - regulation of ppar and ppar expression , while in ppar mice expression of ppar/ was significantly down - regulated ( fig .
these data show that ppars are crucial for maintaining gys-2 expression in fat , although it is difficult to ascertain which ppar isotype is the main regulator of gys-2 expression in wat . 

figure 1expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) .
expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr .
the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) .
( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr .
data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist.table 1genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice.geneproductfold decrease micro - arrayq - pcrglucose metabolismgys2glycogen synthase 213.279.28pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.99pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.64gys3glycogen synthase 3 , brain2.25ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen2.19slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.161.56slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.041.56pfk-26-phosphofructo-2-kinase / fructose-2,6-bisphosphatase2.01gdc1glycerol phosphate dehydrogenase 1 , cytoplasmic adult1.93ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen1.91gdc1glycerolphosphate dehydrogenase 1 , cytoplasmicadult1.84pyglliver glycogen phosphorylase1.71lipid metabolismppargperoxisome proliferator activator receptor gamma2.993.63c5dsterol - c5-desaturase2.85lrp1low density lipoprotein receptor - related protein 12.22cd36cd36 antigen2.203.09]fads3fatty acid desaturase 32.06lipelipase , hormone sensitive1.961.66lrp2low density lipoprotein receptor - related protein 21.91fabp5fatty acid binding protein 5 , epidermal1.87dgat1diacylglycerol acyltransferase1.87decr12,4-dienoyl coa reductase 1 , mitochondrial1.85slc27a1solute carrier family 27 ( fatty acid transporter ) , member 11.85phyhphytanol - coa hydroxylase1.77dgat2/1diacylglycerol o - acyltransferase 2-like 11.75ppargc1peroxisome proliferative activated receptor , gamma , coactivator 11.741.97 expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) .
expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr .
the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) .
( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr .
data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist .
genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice . to investigate whether expression of gys-2 in adipocytes is under direct control of ppars , the effect of ppar ligands on gys-2 mrna was studied in differentiated mouse 3t3-l1 adipocytes .
it was observed that the ppar/ agonist l165041 , and the ppar agonists ciglitazone and rosiglitazone significantly induced gys-2 mrna levels ( fig .
thus , gys-2 may represent a direct target gene of ppar and ppar/ in adipocytes .
it was observed that liver glycogen levels were reduced in refed ppar-/- mice compared to refed wild - type mice [ 30 , 31 ] , which we confirmed using histochemical staining ( fig .
expression of gys-2 is highest in liver , followed by wat ( our unpublished data ) .
we confirm that hepatic gys-2 mrna is markedly reduced in ppar-/- mice ; however , only in the 24-h fasted and refed state ( fig .
3a ) . to further examine the role of ppar in gys-2 expression , primary hepatocytes from wild - type and ppar-/- mice were treated with the synthetic ppar agonist wy14643 , allowing for a direct evaluation of the effect of ppar activation on gys-2 expression .
basal expression of gys-2 was about fourfold reduced in ppar-/- hepatocytes , indicating a requirement for ppar ( fig .
furthermore , wy14643 stimulated gys-2 expression in wild - type but not in ppar-/- hepatocytes ( fig .
3b ) . induction of gys-2 expression by synthetic ppar agonists was also observed in rat primary hepatocytes ( fig .
3c ) . together , these data suggest a direct role of ppar in governing hepatic gys-2 expression . 

figure 2staining for hepatic glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice .
mice were fasted for 24 h followed by refeeding for 7 h before sacrifice.figure 3ppar governs hepatic expression of gys-2 .
( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr .
mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting .
significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr .
significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) .
( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) .
the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) .
glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice .
( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr .
mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting .
significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr .
significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) .
( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) .
the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) .
identification of a putative ppre in the proximal promoter of the mouse gys-2 gene . to determine what genomic region could be responsible for the ppar - induced up - regulation of gys-2 mrna
, the mouse gys-2 gene was scanned for potential ppres ( nubiscan algorithm and hidden markov model framework ) [ 33 , 34 ] .
a direct repeat-1 motif ( dr - lprom ) was localized to the proximal gys-2 gene promoter , about 169 bp upstream from the transcription start site . with the exception of two nucleotides ,
dr - lprom is identical to the consensus sequence , suggesting that this sequence could serve as a functional ppre ( fig .
figure 4the dr-1 present in the gys-2 promoter does not mediate ppar - dependent transactivation .
( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint .
( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone .
chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) .
the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes .
( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint .
( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone .
chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) .
the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes .
( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . to examine whether the promoter region containing the putative ppre is responsible for ppar - dependent up - regulation of gys-2 expression , a 553-nucleotide fragment of the mouse gys-2 promoter gene
was cloned in front of a luciferase reporter gene and transactivation studies were carried out in hepg2 cells .
surprisingly , co - transfection of ppar or pparl expression vectors in combination with ppar agonists slightly decreased luciferase activity , while ppar/ activation had little effect ( fig .
transactivation assays performed with a small genomic fragment surrounding dr - lprom cloned in front of sv40-luciferase led to a similar overall ppar - mediated repression for ppar and pparl , while ppar/ had little effect ( fig .
co - transfection of rxr or of different co - activators such as cbp and pgcl did not change this pattern ( data not shown ) .
thus , the ppre identified in the gys-2 promoter probably does not mediate the effect of ppars on gys-2 expression .
nevertheless , chip experiments carried out in 3t3-l1 cells indicated that ( 1 ) ppar was bound to dr - lprom in mature adipocytes , but not in pre - adipocytes ( fig .
4d ) , and ( 2 ) ppar/ was bound to dr - lprom in pre - adipocytes and , more strongly , in mature adipocytes ( fig .
4e ) . thus , despite dr - lprom behaving poorly as a ppre in classical transactivation assay , it binds both ppar and ppar/ in adipocytes .
this suggests that in vivo binding of ppar and ppar/ to dr - lprom does not translate into transcriptional activation of the gys-2 gene , and accordingly that activation of gys-2 expression by ppars may be mediated by another genomic region .
it should be mentioned that chip did not reveal any binding of ppar to dr - lprom in hepatocytes ( data not shown ) .
interestingly , using the same strategy as described above , a putative ppre that is homologous to the consensus dr-1 sequence was identified in intron 1 of the mouse gys-2 gene ( fig .
4a ) . to assess whether dr - lint was able to mediate ppar - dependent transactivation , a 314-nucleotide genomic fragment surrounding dr - lint was cloned in front of the sv40 promoter followed by a luciferase reporter gene . in hepg2 cells treatment with the synthetic ppar agonist
wy14643 induced reporter activity and this activation was further enhanced upon co - transfection of mppar ( fig .
similar inductions of reporter activity were observed for ppar/ and ppar and their respective agonists ( fig .
thus , dr - lint is able to mediate ppar - dependent transactivation , irrespective of the ppar isotype , suggesting that it may at least be partially responsible for ppar - dependent regulation of gys-2 expression . 

figure 5gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) .
( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone .
( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing
gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein .
fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated .
gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) .
( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors .
luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone .
( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing
gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein .
fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . in agreement with the transactivation data , ppar , ppar/ and ppar proteins
a retarded heterodimeric complex was observed only in the presence of both ppar and obligate binding partner rxr ( fig .
the complex disappeared in the presence of an excess of cold specific oligonucleotide , but not nonspecific oligonucleotide .
examination of in vivo ppar binding to dr - lint by chip yielded very similar results as for dr - lprom : ppar was bound to dr - lint in mature 3t3-l1 adipocytes , but not in pre - adipocytes ( fig .
6a ) , whereas ppar/ was bound to dr - lint in both pre- and mature adipocytes ( fig .
chip analysis demonstrated binding of ppar to dr - lint in wild - type but not ppar mice , and binding was enhanced by fasting and wy14643 ( fig .
together , these data indicate that mouse gys-2 is a direct ppar target gene and that regulation by ppars is at least partially mediated by a ppre present in intron 1 . 

figure 6ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo .
chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) .
the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) .
pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo .
chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) .
the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) .
pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna .
gys-2 is a novel direct target of the liver enriched factor hnf4. as explained above , ppar caused a reduction in gys-2 promoter activity via dr - lprom ( fig .
a similar decrease of promoter activity in response to ppar despite the presence of a putative ppre has been reported for other genes .
indeed , it was found that ppar decreases expression of the apociii and transferrin genes via competition with the hnf4. since hnf4 is known to recognize dr-1 sequences as well , we examined whether hnf4 might control the expression of gys-2 in liver , possibly via dr - lprom .
gys-2 mrna levels were markedly decreased in liver - specific hnf4null mice , thus supporting a role for hnf4 in regulating gys-2 expression ( fig .
7a ) . in transactivation assays using the gys-2 promoter , hnf4 markedly activated reporter activity , suggesting the presence of a hnf4 response element within the 0.55-kb promoter fragment ( fig .
7b ) . mutating dr - lprom resulted in an approximately 50 % reduction in hnf4-dependent activation of the gys-2 promoter ( fig .
7c ) , which suggests that ( a ) hnf4 responsiveness is partially mediated by dr - lprom , or ( b ) the mutations with dr - lprom only partially disabled hnf4 responsiveness .
regardless of these explanations , these data suggest that hnf4 directly regulates the hepatic expression of gys-2 at least partially via dr - lprom .
finally , chip clearly showed hnf4 binding to dr - lprom but not dr - lint in rat primary hepatocytes ( fig .
figure 7gys-2 dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) .
( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes .
( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector .
normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 .
dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) .
( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes .
( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector .
normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 .
whereas hnf4 activates the gys-2 promoter via dr - lprom , ppar does the opposite , suggesting that ppar may interfere with gys-2 promoter activation by hnf4. to examine whether this is the case , the effect of ppar on hnf4-mediated transactivation of the 0.55-kb gys-2 promoter was studied .
ppar activation significantly reduced hnf4-dependent transactivation , indicating competition between hnf4 and ppar in the regulation of the gys-2 promoter ( fig .
as already mentioned above , we failed to find any evidence for binding of ppar to dr - lprom in hepatocytes .
thus , the inhibitory effect of ppar on transcriptional activation of gys-2 by hnf4 likely does not occur via competition with hnf4 for actual binding to dr - lprom .
in the present study , we have identified the mouse gys-2 gene as a direct ppar and hnf4 target gene .
we have shown that the effects of ppars and hnf4 on gys-2 expression occur via two distinct response elements .
indeed , while transcriptional activation of the gys-2 gene by ppars was found to be mediated by a ppre present in intron 1 of the mgys-2 gene ( dr - lint ) , the stimulatory effect of hnf4 was mediated by a response element in the immediate upstream promoter ( dr - lprom ) .
our data are suggestive of the following scenario . in liver , which expresses high amounts of hnf4 , dr - lprom is occupied by hnf4 but not ppar , while dr - lint is bound by ppar but not hnf4. hence , hnf4 and ppar activate gys-2 expression via different response elements . nevertheless , important negative cross - talk between the two nuclear receptors was observed . in the absence of any mutual binding to the response elements , it can be hypothesized that competition may take place at the level of binding to common co - activator proteins in a mechanism that is often referred to as squelching . in adipose tissue , which does not express hnf4 , dr - lprom
is occupied by ppar/ and ppar , but this does not result in transcriptional activation . rather , transactivation occurs via binding of ppar/ and ppar to dr - lint .
our data indicate that hnf4 is an extremely powerful activator of mouse gys2 transcription , explaining the marked reduction in hepatic gys-2 expression in liver - specific hnf4-null mice . as mentioned above
, regulation of mgys-2 expression by hnf4 at least partially occurs via dr - lprom .
a recent study that combined chip with promoter micro - arrays showed that the gys-2 promoter is bound by hnf4 in human liver , thus establishing gys-2 as a direct target of hnf4 in human as well .
it is not very clear why disabling the dr - lprom reduced hnf4-dependent trans - activation by only 50 % .
it is possible that the 0.55-kb gys-2 promoter fragment contains an additional hnf4 response element , although in silico analysis failed to reveal such an element .
alternatively , it is possible that mutating the wild - type dr - lprom ( aggccaaaggcca ) into a mutated dr - lprom ( aggcctttggcca ) only partially disabled the response element . while functional ppres are commonly located within regulatory sequences , i.e. , proximal promoters , ppres have also been identified in intronic sequences .
examples are ppres within intron 3 of human / mouse angptl4 and rat peroxisomal thiolase b genes , and within intron 1 of the rat acyl - coa binding protein gene and human carnitine palmitoyltransferase 1a [ 25 ] .
our data demonstrate that regulation of gys-2 expression by ppars is also mediated by an intronic ppre . in the past few years , our understanding of the function of ppar/ in vivo has improved greatly thanks to studies using various transgenic mouse models [ 19 , 37 ] . at the level of metabolism , ppar/ over - expression promotes skeletal muscle fatty acid oxidation and type i fiber content in mice , resulting in improved endurance exercise performance .
conversely , deletion of ppar/ in cardiomyocytes is associated with impaired fatty acid oxidation and expression of fatty acid oxidative genes , whereas glucose uptake is increased . in wat , ppar/ stimulates fatty acid oxidation and uncoupling , thereby diminishing adiposity .
it is thus clear that ppar/ plays a pivotal role in governing fatty acid oxidation in a variety of tissues . in contrast
, data linking ppar/ to regulation of glucose homeostasis remain scarce [ 20 , 39 , 40 ] .
our data reveal that ppar/ is a critical regulator of the adipose expression of the gys-2 gene . furthermore , micro - array and qpcr analysis indicated that expression of numerous other genes involved in lipid and glucose metabolism was markedly down - regulated in ppar/-null mice , including glut4 , p85 , and cd36 . since ppar and pgc - l
were significantly down - regulated as well , it is possible that many of the observed changes are not linked to the absence of ppar/ per se but rather reflect indirect effects mediated via decreased ppar and pgc - l mrna .
although such an effect may contribute to some extent to the down - regulation of gys-2 in ppar/-null mice , the in vitro studies leave no doubt that gys-2 is a direct target gene of ppar/ , as well as of ppar. glycogen is stored in many tissues , yet it is particularly abundant in liver , muscle , and adipose tissue . in liver , glycogen serves to maintain blood glucose levels between meals , while skeletal muscle glycogen is used to fuel muscle contractions .
in contrast , adipose tissue glycogen serves as a source of glycerol 3-phosphate , which is required for ( re)-esterification of fatty acids into triglycerides .
several alternative pathways exist to produce glycerol 3-phosphate , including synthesis from glucose , and conversion of gluconeogenic precursors ( glyceroneogenesis ) . since expression and activity of glycerol kinase are very low in adipose tissue , direct phosphorylation of glycerol is not considered as a major pathway to generate glycerol 3-phosphate .
however , recent studies suggest that this may change after treatment with synthetic ppar agonists , which markedly up - regulate glycerol kinase expression in human and mouse adipocytes [ 11 , 42 ] .
in fact , it has been hypothesized that stimulation of glycerol kinase expression by tzds , resulting in increased fatty acid re - esterification , may at least partially account for the suppressive effect of tzds on plasma free fatty acid levels .
stimulation of fatty acid esterification is part of a general lipogenic and adipogenic effect of ppar in the adipocyte .
since adipose glycogen stores yield glycerol 3-phosphate as a precursor for fatty acid ( re-)esterification , up - regulation of gys-2 expression by ppar can be placed in the context of the lipogenic role of ppar in the adipocyte , which is aimed at promoting energy storage . besides contributing to lipogenesis , synthesis of glycogen permits continued uptake of glucose into cells .
accordingly , it can be speculated that up - regulation of adipose gys-2 by ppar might partially account for the stimulation of glucose uptake into adipocytes by ppar agonists .
it is currently still ambiguous whether ppar/ serves a general anabolic or catabolic function in the adipocyte . on the one hand
, it has been reported that ppar/ promotes fatty acid oxidation in adipocytes [ 20 , 21 ] .
on the other hand , ppar/ also seems to have a facilitative , yet important role in lipo- and adipogenesis .
as discussed above for ppar , up - regulation of gys-2 expression by ppar/ may indicate a role for ppar/ in fatty acid ( re-)esterification , thus contributing to a lipogenic role for ppar/. the highest levels of glycogen are found in liver and fluctuate with nutritional status .
remarkably , expression of gys-2 in liver increases during fasting , at the same time when glycogen stores are actively broken down .
the reason behind this seemingly counterintuitive regulation is not very clear , but it may serve to prime the glucose synthesizing system for when dietary glucose becomes available again . in the absence of ppar
, we observed that the expression of gys-2 drops markedly during prolonged fasting and refeeding .
the reduced gys-2 expression is likely responsible for the diminished rate of glycogen formation upon refeeding , as observed by us and previously by others .
indeed , the effect of ppar deletion on liver glycogen is minor except under conditions of refeeding [ 3032 ] .
it has been reported that after a short - term fast the gluconeogenic flux in ppar-null mice is directed more towards glycogen , leading to a decrease in hepatic glucose output .
however , it is unclear what happens to the gluconeogenic flux toward glycogen in the fasted - refed state , although our and other data clearly indicate that total glycogen synthesis is decreased in ppar-/- mice .
mutation of the gys2 gene in humans leads to lower hepatic glycogen levels and fasting hypoglycemia , biochemical features that are also observed in ppar-/- animals .
opposite to that observed in patients with a dysfunctional gys2 gene , ppar-/- mice show low plasma ketones , which is explained by the stimulatory effect of ppar on fatty acid oxidation and ketogenesis .
overall , our data suggest that the decreased hepatic glycogen levels in ppar-/- and liver - specific hnf4-null mice [ 28 , 35 ] may be due to decreased activation of gys-2 expression via dr - lint and dr - lprom , respectively .
although ppar and hnf4 stimulate gys-2 expression via different response elements , important interplay exist between signaling of the two nuclear receptors . in conclusion , we show that gys-2 is a direct target gene of ppars .
 abstract.glycogen synthase 2 ( gys-2 ) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue , yet little is known about regulation of gys-2 transcription . 
 the peroxisome proliferator - activated receptors ( ppars ) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well . here , we show that gys-2 is a direct target gene of ppar , ppar/ and ppar. expression of gys-2 is significantly reduced in adipose tissue of ppar-/- , ppar/-/- and ppar+/- mice . 
 furthermore , synthetic ppar/ , and  agonists markedly up - regulate gys-2 mrna and protein expression in mouse 3t3-l1 adipocytes . in liver , 
 ppar deletion leads to decreased glycogen levels in the refed state , which is paralleled by decreased expression of gys-2 in fasted and refed state . 
 two putative ppar response elements ( ppres ) were identified in the mouse gys-2 gene : one in the upstream promoter ( dr-1prom ) and one in intron 1 ( dr-1int ) . 
 it is shown that dr-1int is the response element for ppars , while dr-1prom is the response element for hepatic nuclear factor 4 alpha ( hnf4 ) . in adipose tissue , which does not express hnf4 , dr-1prom 
 is occupied by ppar/ and ppar , yet binding does not translate into transcriptional activation of gys-2 . 
 overall , we conclude that mouse gys-2 is a novel ppar target gene and that transactivation by ppars and hnf4 is mediated by two distinct response elements . 
Introduction
Materials and methods
Results
Expression of Gys-2 in WAT is regulated by PPARs.
Discussion