in healthy humans , skeletal muscle accounts for ~7080% of the insulin - stimulated glucose uptake , being the major site of glucose disposal and , thus , exerting a key role in regulating whole body glucose homeostasis . accordingly , understanding changes that occur to this tissue during obesity and diabetes development is crucial to elucidate the underlying causes of insulin resistance and to reveal new targets for its treatment . insulin resistance in skeletal muscle
has long been recognized as a characteristic feature of type 2 diabetes and plays a major role in the pathogenesis of the disease .
although several epidemiological data have shown that the consumption of added sugars as ingredients in processed or prepared foods and caloric beverages has dramatically increased over the last decades , most of the experimental studies investigating the development of insulin resistance in the skeletal muscle have been based on genetic manipulation or use of high fat diets [ 24 ] .
in contrast , the molecular mechanisms underlying the detrimental effects of sugar , mainly those on skeletal muscle , are not completely understood . in the present study , we used a previously developed mouse model of chronic exposure to high - fructose corn syrup ( hfcs )   to investigate the deleterious effects of high sugar intake on skeletal muscle .
hfcs syrup , used as an ingredient in processed or prepared foods and caloric beverages , is synthesized by refining corn starch , contains 55% fructose and 42% glucose and to date accounts for over 40% of all added caloric sweeteners .   the peroxisome proliferator activated receptor ( ppar ) superfamily of transcription factors , that includes the isoforms ppar- , ppar- , and ppar- , has been widely shown to exert crucial roles in energy homeostasis regulation and glucose metabolism .
ppar- , one of the most promising pharmacological target implicated in obesity - associated insulin resistance , is highly expressed in skeletal muscle , at 10- and 50-folds higher levels compared with ppar- and ppar- , respectively .
however , its potential effects in affecting skeletal muscle glucose intake and insulin sensitivity are only now being elucidated .
schuler et al .   showed that mice in which ppar- is selectively ablated in skeletal myocytes exhibit fiber - type switching , obesity , and type 2 diabetes .
besides , a very recent paper has suggested that the improvement of glucose homeostasis by angiotensin receptor blockers in hypertensive patients involves a selective ppar- activation in the skeletal muscle .
so far , the exact molecular mechanisms underlying the observed ppar--induced changes have not yet been determined .
hence , the present study aimed to determine whether chronic administration of the selective ppar- agonist gw0742 in hfcs - fed mice may ameliorate the impairment of signaling pathways triggered in skeletal muscles by chronic high sugar intake .
several studies have revealed that a large number of muscle - derived secretory cytokines ( collectively termed myokines ) can act locally in an autocrine / paracrine manner , linking skeletal muscle to regulation of physiological processes in other tissues .
we have , thus , also investigated whether ppar- agonism may affect the serum levels of the well - known myokine interleukin-6 ( il-6 ) and the muscular expression of a member of the fibroblast growth factor ( fgf ) superfamily , fgf-21 , a recently identified myokine involved in the interorgan communication . to further extend our investigation on the ability of ppar- agonism to modulate inflammatory pathways involved in local insulin resistance pathogenesis ,
the effects of ppar- activation on nuclear translocation of the transcription nuclear factor - kappab ( nf-b ) and expression of its target genes have also been studied .
four - week - old male c57bl6/j mice ( harlan - italy ; udine , italy ) were housed in a controlled environment at 25  2c with alternating 12-h light and dark cycles .
they were provided with a piccioni pellet diet ( n. 48 , gessate milanese , italy ) and water ad libitum .
all the animals were fed with a normal pellet diet for 1 week prior to the experiment .
the animals were then allocated to two dietary regimens , chow diet and normal drinking water ( control ) or a chow diet and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs ) for 30 weeks .
the concentration of hfcs solution as well as the period of dietary manipulation was chosen according to previous animal studies investigating the metabolic effects of long - term ( 6 - 7 months ) access to hfcs .
animal care was in compliance with italian regulations on the protection of animals used for experimental and other scientific purposes ( dm 116/92 ) , and the experiment was approved by the turin university ethics committee . after the initial period of 14 weeks of dietary manipulation , each of the two diet groups ( control and hfcs diet ) was further subdivided to obtain four different treatment groups : chow diet and normal drinking water ( control , n = 10 ) , chow diet supplemented with gw0742 ( 1  mg / kg / day ) and normal drinking water ( control + gw , n = 6 ) , chow diet and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs , n = 10 ) , and chow diet supplemented with gw0742 ( 1  mg / kg / day ) and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs + gw , n = 10 ) . the drug was daily administered with the food for the last 16 weeks , and the mice were allowed to continue to feed on their respective diets until the end of the study .
gw0742 is a highly potent and selective ppar- agonist ( murine ec50 : 28  nm for ppar- ; 8,900  nm for ppar- ; > 10,000  nm for ppar- ) , with an acceptable pharmacokinetic profile and activity in vivo .
the dose and the kinetics of administration were chosen based on those we have previously shown to improve glucose tolerance and insulin sensitivity in vivo .  
one day before the mice were due to be killed , the ogtt was performed after a fasting period of 6 h by administering glucose ( 2  g / kg ) by oral gavage . once before administration and 15 , 30 , 60 , 90 , 120 , and 150  min afterward
, blood was obtained from the saphenous vein , and glucose concentration was measured with a conventional glucometer ( accu - check compact kit , roche diagnostics gmbh , mannheim , germany ) .  
after 16 weeks from the start of the drug treatment ( i.e. , after 30 weeks of dietary manipulation ) , the mice were anaesthetised with i.p .
injection ( 30  mg / kg ) of zoletil 100 ( laboratories virbac , france ) and killed by aortic exsanguination .
the serum lipid profile was determined by measuring the content of triglycerides , total cholesterol , high - density lipoprotein ( hdl ) , and low density lipoprotein ( ldl ) by standard enzymatic procedures using reagent kits ( hospitex diagnostics , florence , italy ) .
plasma leptin and adiponectin levels were measured using enzyme - linked immunosorbent assay ( elisa ) kits ( leptin and adiponectin mouse elisa kits , abcam , cambridge uk ) .
the gastrocnemius muscle and epididymal fat were isolated , weighed , rapidly freeze - clamped with liquid nitrogen , and stored at 80c .
briefly , gastrocnemius were homogenised at 10%(w / v ) in a potter - elvehjem homogenizer ( wheaton , nj , usa ) using a homogenisation buffer containing 20  mm hepes , ph 7.9 , 1  mm mgcl2 , 0.5  mm edta , 1  mm egta , 1  mm dithiothreitol ( dtt ) , 0.5  mm phenylmethylsulfonyl fluoride ( pmsf ) , 5  g / ml aprotinin , and 2.5  g / ml leupeptin .
supernatants were removed and centrifuged at 14000  rpm at 4c for 40 minutes to obtain the cytosolic fraction .
the pelleted nuclei were resuspended in an extraction buffer and centrifuged at 14000  rpm for 20 minutes at 4c .
the amount of proteins contained in the cytosolic and nuclear fraction was determined using a bca protein assay following the manufacturers ' instructions .
skeletal muscle triglycerides were extracted from gastrocnemius homogenates and assayed using reagent kits according to the manufacturer 's instructions ( triglyceride quantification kit , abnova corporation , aachen , germany ) . about 60  g of total proteins was loaded for western blot experiments .
proteins were separated by 8% sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to a polyvinyldene difluoride ( pvdf ) membrane , which was then incubated with a primary antibody ( rabbit anti - ppar- , dilution 1  :  500 ; rabbit antitotal gsk-3 , dilution 1  :  200 ; goat anti - pgsk-3 ser , dilution 1  :  200 ; rabbit antitotal akt , dilution 1  :  1000 ; mouse anti - pakt ser , dilution 1  :  1000 ; goat anti - icam-1 , dilution 1  :  500 ; rabbit antitotal irs-1 , dilution 1  :  200 ; goat anti - pirs-1 ser , dilution 1  :  200 ; rabbit anti - glut-4 , dilution 1  :  2000 ; rabbit anti - glut-5 , dilution 1  :  100 ; rabbit anti - inos , dilution 1  :  200 ; rabbit anti - nf-b dilution 1  :  1000 ; rabbit antitotal ampk , dilution 1  :  1000 ; rabbit anti - pampk thr , dilution 1  :  1000 ; rabbit antitotal acc , dilution 1  :  500 ; rabbit anti - pacc ser , dilution 1  :  1000 ; rabbit anti - cpt-1 , dilution 1  :  200 ) .
blots were then incubated with a secondary antibody conjugated with horseradish peroxidase ( dilution 1  :  10000 ) and developed using the ecl detection system .
the immunoreactive bands were visualised by autoradiography and the density of the bands was evaluated densitometrically using gel - pro analyzer 4.5 , 2000 software ( media cybernetics , silver spring , md , usa ) .
the membranes were stripped and incubated with tubulin monoclonal antibody ( dilution 1  :  5000 ) and subsequently with an anti - mouse antibody ( dilution 1  :  10000 ) to assess gel - loading homogeneity .
for immunodetection of glucose transporter type-4 ( glut-4 ) , sections were incubated overnight with rabbit anti - glut-4 antibody ( abcam ) , dilution 1  :  200 , and for 1 hour with goat anti - rabbit igg - hrp conjugated secondary antibody ( bio - rad laboratories , hercules , ca , usa ) . for detection of glucose transporter type 5 ( glut-5 )
, sections were incubated overnight with rabbit anti - glut-5 antibody ( abcam ) , dilution 1  :  50 , for 1 hour with swine anti - rabbit igg - biotinylated secondary antibody ( dako , glostrup , denmark ) , and for 1 hour with streptavidin hrp conjugate ( southern biotech , birmingham , al , usa ) .
the specific staining was detected with diaminobenzidine ( dab , sigma - aldrich ) , and sections were visualized with olympus - bx4i microscope connected by a photographic attachment ( carl zeiss , oberkochen , germany ) .
for each antibody , a negative control was included in which the primary antibody was replaced with a nonimmune isotypic control antibody .
serum levels of il-6 and fgf-21 levels in gastrocnemius homogenates were measured by elisa according to the manufacturer 's instructions ( mouse fgf-21 and il-6 elisa kits , r&d systems , abingdon , uk ) . unless otherwise stated
( rockford , il , usa ) , and pvdf was from the millipore corporation ( bedford , massachusetts , usa ) .
antibodies were from cell - signaling technology ( beverly , ma , usa ) , santa cruz biotechnology ( santa cruz , ca , usa ) , and abcam ( cambridge , cb , uk ) . the anti - mouse , anti - rabbit , and anti - goat horseradish peroxidase - linked antibodies were from santa cruz biotechnology ( santa cruz , ca , usa ) , and luminol ecl was from perkinelmer ( waltham massachusetts , usa ) .  
all values in both the text and figures are expressed as mean  sd for n observations .
one - way analysis of variance with dunnett 's post - hoc test was performed using the graphpad prism version 4.02 for windows ( graphpad software , san diego , california , usa ) , and p values below 0.05 were considered as significant .
after 30 weeks of dietary manipulation , mice on hfcs group had significantly higher body mass than the control group , with an increase in body mass over 30% ( 36.00  1.41  g versus 32.25  1.67  g ; p
< 0.01 ) , whereas gw0742 administration induced a slight but not significant reduction in body mass ( 35.00  2.83  g ) .
epididymal fat mass was increased by hfcs manipulation in comparison to control diet ( 4.22  0.35% body mass versus 3.66  0.29% body mass ; p < 0.01 ) , and values were reduced to the control level by drug administration ( 3.86  0.33% body mass ) .
in contrast , neither dietary manipulation nor drug treatment affected gastrocnemius mass ( % body mass : 0.97  0.19 control , 0.92  0.09 hfcs , and 0.92  0.06  hfcs + gw ) . as we previously reported , the hfcs diet caused a significant increase in serum triglycerides , total cholesterol , and ldl concentrations and a marked decrease in the hdl levels .
interestingly , the hfcs diet significantly affected insulin sensitivity . a significant increase in serum insulin levels
was observed in mice of hfcs group compared to control mice ( 2.26  0.51  g / l versus 1.27  0.07 
g / l ) , and this increase was almost completely abolished by gw0742 ( 1.51  0.46  g / l ) .
fasting glucose concentrations were elevated in serum of hfcs animals in comparison to control animals ( 103.4  12.0  mg / dl versus 78.7  0.3  mg / dl ) and reduced to control levels by gw0742 ( 81.3  10.9  mg / dl ) .
moreover , hfcs mice showed a significant impairment in glucose tolerance to exogenously administered glucose .
although all the groups reached a glycemic peak at 15  min postglucose challenge ( control : 223.1  12.0  mg / dl ; hfcs : 212.7  7.3  mg / dl ; hfcs + gw : 198.8  10.3  mg / dl ) , glycemic levels at 3060  min post glucose challenge in hfcs - fed mice were higher than those recorded in the control group ( 30  min : 182.9  8.3  mg / dl versus 129.4  8.0  mg / dl , p < 0.05 ; 60  min : 130.7  10.3  mg / dl versus 96.8  4.0  mg / dl , p < 0.05 ) .
gw0742 significantly ( p < 0.05 ) improved glucose tolerance in hfcs - fed mice , showing significantly reduced glucose concentrations at both 30 and 60  min postglucose challenge ( 89.7  5.2  mg / dl and 86.7  4.3  mg / dl , resp . ) .
data on the effects of dietary manipulation and chronic gw0742 treatment on serum levels of il-6 , adiponectin , and leptin are reported in table 1 .
the experimental diet caused a more than fourfold increase in serum il-6 concentrations , whereas gw0742 administration significantly decreased the il-6 levels .
adiponectin and leptin showed different patterns : in fact , the level of adiponectin in hfcs mice was lower than in control mice , while that of leptin increased versus controls .
when gw0742 was administered to hfcs mice there was a significant increase ( p < 0.01 ) in the adiponectin levels associated with a significant decrease ( p < 0.01 ) in leptin serum concentration .  
it must be noted that in control mice gw0742 had no significant effect on any of the previously described metabolic parameters . as shown in figure 1
in contrast , daily administration of gw0742 resulted in a twofold increase in expression of its pharmacological target , with maximum effect in the presence of dietary manipulation .  
the hfcs diet did not alter the protein expression of irs-1 , akt , or gsk-3 compared to the control group .
however , hfcs caused a marked increase in ser phosphorylation of irs-1 in parallel with reduced ser phosphorylation of akt ( figures 2(a ) and 2(b ) ) .
ser phosphorylation of gsk-3 , a downstream target of akt , was also reduced in the presence of hfcs ( figure 2(c ) ) , suggestive of impaired insulin signaling downstream of irs-1 .
most notably , gw0742 significantly attenuated all the effects of hfcs on irs-1 , akt , and gsk-3 phosphorylation , measured at the steady state . in comparison to control animals ,
glut-4 expression was reduced in the gastrocnemius of hfcs mice , without reaching a statistical significance , and increased following drug treatment ( figure 3(a ) ) .
similarly , the hfcs diet markedly reduced glut-5 expression , whereas gw0742 administration resulted in a significant increase in glut-5 expression ( figure 3(c ) ) .
notably , gw0742 treatment not only increased carriers expression levels but also induced a significant membrane translocation , thus increasing muscle glucose uptake ( figures 3(b ) and 3(d ) ) .
the triglyceride content was doubled in the gastrocnemius of hfcs mice in comparison with control animals , whereas skeletal muscle triglyceride accumulation was significantly reduced by gw0742 administration ( figure 4 ) .  
changes in the phosphorylation / activation of the amp - activated protein kinase ( ampk)/acetyl - coa carboxylase ( acc ) system , whose central role in the regulation of cellular lipid homeostasis is well known , were evaluated by immunoblotting experiments on gastrocnemius homogenates .
as reported in figure 5 , there was no significant effect of hfcs feeding or drug treatment on total ampk and acc protein content .
in contrast , chronic hfcs exposure markedly reduced thr phosphorylation of ampk and its substrate ser acc .
interestingly , gw0742 treatment led to a robust increase in phosphorylation of both ampk and acc in hfcs - fed mice ( figures 5(a ) and 5(b ) ) .
the effects of ppar- agonism on fatty acid oxidation in skeletal muscle cells are further indicated by the significant increase in the protein expression of carnitine palmitoyl transferase-1 ( cpt-1 ) elicited by gw0742 in hfcs - fed mice ( figure 5(c ) ) . to investigate the muscle inflammatory response induced by hfcs - diet and the intracellular signaling pathway(s ) that might be involved in the protective mechanisms evoked by gw0742
when compared to control mice , hfcs mice developed significant increase in the nuclear translocation of the p65 nf-b subunit in the mouse gastrocnemius , indicating the activation of this transcriptional factor ( figure 6(a ) ) .
treatment with gw0742 resulted in a significant reduction in nuclear translocation of p65 and , hence , in the activation of nf-b .
the intercellular adhesion molecule-1 ( icam-1 ) , whose role in the recruitment of neutrophils is widely accepted , was slightly detected in the gastrocnemius of control animals , whereas its expression was dramatically increased by hfcs diet ( figure 6(b ) ) .
as shown in figure 6(c ) , also the expression of the inducible nitric oxide synthase ( inos ) was increased in the presence of dietary manipulation .
interestingly , these changes were suppressed by gw0742 administration , as evidenced by densitometric analysis of the related autoradiograms .
the beneficial effects of ppar- activation were associated with a dramatic increase of fgf-21 production in the mouse gastrocnemius .
as shown in figure 7 , in comparison to control mice ( 46.45  1.13  pg / mg protein ) , hfcs diet did not affect the production of this myokine ( 49.38  4.12  pg / mg protein ) , whereas gw0742 treatment induced a twofold increase ( 87.08  6.37  pg / mg protein ) .
in agreement with previous observations [ 14 , 15 ] , the present study shows that chronic exposure to the most widely used added sugar hfcs caused a significant increase in body mass associated with increases in serum levels of triglycerides , ldl - cholesterol , glucose , insulin , il-6 , leptin , and hypoadiponectinemia . using the same experimental protocol here reported , we have recently demonstrated that the molecular mechanism underlying the deleterious effects of hfcs involves the hepatic upregulation of fructokinase , the main fructose - metabolizing enzyme , which may account for the increase in serum levels of free fatty acids and hyperuricemia .
we also reported that the documented increase in serum uric acid level significantly contributed to the development of chronic kidney injury .
interestingly , we demonstrated that chronic administration of the ppar- ligand gw0742 exerted beneficial effects by preventing the upregulation of fructokinase in the liver and activation of the inflammatory signaling complex nlrp3 inflammasome in the kidney . despite these preliminary data , so far , there are no studies on the effects of long - term exposure to hfcs , the major sweetener added to beverages and food , on skeletal muscle , which is a major site of postprandial glucose disposal and is therefore one of the insulin - sensitive tissues most likely to manifest early signs of insulin resistance . here , we investigated ( i ) the mechanisms underlying metabolic disturbances in skeletal muscle of mice exposed for 30 weeks to high intake of hfcs and ( ii ) the local effects evoked by ppar- chronic activation .
the sugar was added to the drink water at a concentration that covers 10% of the daily caloric intake , corresponding to the average energy intake in the form of ingested sweeteners in the western diet .
we documented the hfcs - induced alteration in the insulin signal transduction pathway , as shown by the impaired phosphorylation of irs-1 protein as well as of the downstream key insulin signaling molecules , akt , and gsk-3 , an akt substrate .
this is in keeping with previous studies showing that irs-1 serine phosphorylation can interfere with subsequent akt and gsk-3 phosphorylation , disrupting insulin signal transduction . in our experimental model , oral administration of the selective ppar- agonist gw0742
was associated with a significant improvement of the defective insulin signaling in the skeletal muscle , which may account at least in part for the changes in serum lipid profile and insulin sensitivity .
as the inhibition of gsk-3 evokes an increase in the glycogen synthesis [ 18 , 19 ] , we may speculate that gw0742 administration modulates muscle glycogen storage through inactivation of gsk-3. although no direct evidence of drug treatment on glucose storage has been reported in our study , we documented the effects of chronic ppar- activation on glucose transporters expression and distribution .
specifically , we found that ppar- activation by gw0742 evoked an increase in expression of glut-4 , the most abundant glucose transporter isoform in skeletal muscle , and its translocation from intracellular compartments to the plasma membrane , thus facilitating glucose transport .
similarly , glut-5 , the fructose carrier with low capacity to transport glucose , was scarcely detectable in the gastrocnemius muscle of hfcs - fed mice , and its expression and membrane translocation were enhanced by chronic ppar- activation . because fructose is highly present in the hfcs diet and contributes to carbohydrate metabolism in muscle , the increase in glut-5 may favor a better utilization of fructose supplied in the diet . in any case , the reduction in glut-4 and glut-5 expression and translocation detected in the hfcs group could represent an adaptation to the chronic exposure to a sugar - enriched diet , while the increases in these carrier isoforms in the presence of the selective ppar- agonist gw0742 may account , at least in part , for the improvement in insulin - induced muscle glucose uptake .
recent data have shown that akt inhibitors or dominant - negative akt constructs regulate the translocation , targeting , and fusion of glut-4-containing vesicles [ 23 , 24 ] .
similarly , irs-1 phosphorylation has been reported to affect glut-4 translocation and subsequent glucose uptake in mouse skeletal myocytes .
we may , thus , speculate that the beneficial effects of the ppar- ligand gw0742 are secondary to activation of the irs-1/akt / gsk-3 pathway .
this is also supported by previous findings showing that the chronic administration of gw0742 in rats results in phosphoinositide - dependent kinase phosphorylation and , hence , increased akt phosphorylation .
another enzyme complex which could be involved in ppar- metabolic effects within the skeletal muscle is ampk , a serine / threonine protein kinase that has emerged as a key player in both lipid and glucose metabolism in skeletal muscles [ 27 , 28 ] . in mouse
c2c12 myotubes , ampk activation potentiated insulin action by reducing irs-1 serine phosphorylation , while reduced muscle ampk activity has been reported to aggravate muscle insulin resistance following a high - fat diet over 30 weeks . in agreement with these observations
, we found that gw0742 increased phosphorylation of thr regulatory site on ampk which , in turn , causes acc phosphorylation .
when acc is inactive ( phosphorylated form ) , a fall in malonyl - coenzyme a occurs , which disinhibits cpt-1 and increases mitochondrial import and oxidation of long - chain fatty acids .
our data demonstrate that chronic exposure to gw0742 administration increased skeletal muscle cpt-1 expression , thus shunting toward fatty acid oxidation , and this effect was associated with reduced triglyceride accumulation .
these findings suggest that the beneficial effects evoked by the ppar- agonist gw0742 are at least partially dependent on ampk activation .
the direct involvement of ppar- agonism in mediating improvements in muscle insulin sensitivity and lipid metabolism is also confirmed by results showing that ppar- is expressed in the gastrocnemius of both control and hfcs - fed mice and , more importantly , that its expression is substantially increased by chronic administration of gw0742 , thus suggesting that the observed drug effects are due to a ligand - dependent ppar- activation .
this is consistent with previous works showing that ppar- activation is involved in regulating myogenesis [ 3235 ] . in search of the mechanism(s ) underlying the protective action of gw0742 , we investigated whether ppar- activation may affect the local inflammatory response associated with muscular metabolic injury .
although ppar- has been implicated in the regulation of systemic inflammatory responses associated with metabolic dysregulation , so far , reports on anti - inflammatory effects of ppar activation in skeletal muscle are rather scarce . here
, we show for the first time that in the skeletal muscle gw0742 reverts the diet - induced increase in the nuclear translocation of nf-b p65 , a transcriptional factor that plays an important role in regulating the transcription of a number of genes , especially those involved in producing mediators of local and systemic inflammation ( such as cytokines , chemokines , and cell adhesion molecules ) .
this observation is in agreement with previous studies reporting that ppar- activation induces the physical interaction between ppar- and the p65 subunit of nf-b and reduced the lps - induced degradation of the inhibitory protein 
inhibitor of kappa b ,  thus preventing nf-b activation [ 36 , 37 ] .
the reduction of nf-b activation by gw0742 treatment may account for the observed reduction in the serum levels of il-6 , which is known to be mostly released from skeletal muscle , and in the local expression of the nf-b - dependent proteins inos and icam-1 , whose role in the development of insulin resistance has been recently documented [ 38 , 39 ] .
as several studies have reported an association between gsk-3 and nf-b activity , we might speculate that ppar- activation phosphorylates , and hence , activates the akt pathway , which in turn phosphorylates , and hence , inhibits gsk-3 , presumably resulting in the inhibition of nf-b and , in turn , nf-b - dependent proinflammatory gene transcription . although this cross talk among different signaling pathways by pharmacological ppar- modulation is intriguing , it must be stressed , however , that the lack of direct experimental evidence of a causal relationship between the improved local and systemic insulin sensitivity and the treatment - induced reduction of nf-b activation in our experimental model limit the interpretation of the molecular mechanism(s ) underlying our findings . one of the most recently identified mediators that facilitates organ cross talk and the related control of impaired glucose homeostasis in metabolic diseases is a member of the fgf family , fgf-21 .
fgf-21 has been previously shown to lower blood glucose levels in several diabetic rodent and monkey models [ 41 , 42 ] , to regulate lypolisis in white adipose tissue [ 43 , 44 ] and substrate utilization in the liver .
the metabolic effects of fgf-21 on glucose metabolism involve selective regulation by different ppar isoforms .
conversely , ppar- activation with feeding promotes fgf-21 production in adipocytes but not in liver [ 48 , 49 ] . to our knowledge
, this is the first paper that demonstrates a correlation between ppar- activation and increased fgf-21 levels in skeletal muscle , thus adding an original piece of evidence to the complex mechanisms by which ppar- can regulate several biological functions .
our observation is also in keeping with a previous study showing that circulating fgf-21 levels consistently increase in human subjects in response to pharmacological activation of ppar- .
although a previous investigation has revealed that ppar- directly regulates expression of the fgf-21 gene through elements located within the 500-bp upstream region of the gene , to date , a consensus sequence of ppar- binding site in the promoter region of the fgf-21 gene has not yet been identified .
besides in vitro experiments with fgf-21 , small interfering rna are warranted to clarify whether gw0742 beneficial effects are related to the direct modulation of fgf-21 biological functions .
the recent findings on fgf-21 ability to prevent insulin resistance in human myoblasts by inhibiting nf-b activation   may also suggest that the muscle - derived fgf-21 acts in an autocrine fashion to amplify ppar- inhibitory effects on the expression of nf-b - dependent inflammatory genes .
this further supports the existence of multiple anti - inflammatory pathways involved in the beneficial effects evoked by ppar- activation .   in conclusion
, we have shown that feeding mice with a hfcs diet for 30 weeks evoked skeletal muscle insulin resistance and lipid accumulation which were associated with activation of inflammatory pathways .
in addition , this study , showing a strong induction of fgf-21 in the skeletal muscle after chronic administration of the ppar- ligand gw0742 , enhances our knowledge of the mechanisms of action of ppar- agonism and provides further insights into the role of fgf-21 as mediator of the tissue cross - talk that underlines the integrated control of the metabolic inflammation .
 peroxisome proliferator activated receptor ( ppar)- agonists may serve for treating metabolic diseases . 
 however , the effects of ppar- agonism within the skeletal muscle , which plays a key role in whole - body glucose metabolism , remain unclear . this study aimed to investigate the signaling pathways activated in the gastrocnemius muscle by chronic administration of the selective ppar- agonist , gw0742 ( 1  mg / kg / day for 16 weeks ) , in male c57bl6/j mice treated for 30 weeks with high - fructose corn syrup ( hfcs ) , the major sweetener in foods and soft - drinks ( 15%  wt / vol in drinking water ) . 
 mice fed with the hfcs diet exhibited hyperlipidemia , hyperinsulinemia , hyperleptinemia , and hypoadiponectinemia . in the gastrocnemius muscle , hfcs impaired insulin and amp - activated protein kinase signaling pathways and reduced glut-4 and glut-5 expression and membrane translocation . 
 gw0742 administration induced ppar- upregulation and improvement in glucose and lipid metabolism . 
 diet - induced activation of nuclear factor-b and expression of inducible - nitric - oxide - synthase and intercellular - adhesion - molecule-1 were attenuated by drug treatment . 
 these effects were accompanied by reduction in the serum concentration of interleukin-6 and increase in muscular expression of fibroblast growth factor-21 . 
 overall , here we show that ppar- activation protects the skeletal muscle against the metabolic abnormalities caused by chronic hfcs exposure by affecting multiple levels of the insulin and inflammatory cascades . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion