antibodies for p - ampk ( thr ) , p - stat3 ( tyr ) camp - responsive element binding protein ( p - creb ; ser ) , and phosphorylated glycogen synthase kinase 3 ( p - gsk3 ; ser ) were obtained from cell signaling ( danvers , ma ) ; phosphorylated acetyl coa carboxylase ( p - acc ; ser ) from upstate biotechnologies ( charlottesville , va ) ; total gsk3 from bd transduction laboratories ( san jose , ca ) ; and uncoupling protein 3 ( ucp3 ) from chemicon ( billerica , ma ) . activated receptor- coactivator 1 ( pgc1 ) antibody and 25 dideoxyadenosine were purchased from calbiochem ( san diego , ca ) , rat recombinant il-6 from pierce biotechnologies ( rockford , il ) , and protein a / g plus agarose beads from santa cruz biotechnology ( santa cruz , ca ) . sams  peptide and polyclonal antibodies that immunoprecipitate the 1 or the 2 catalytic subunit of ampk and were used for activity assays were obtained from qcb biotechnology ( hopkinton , ma ) . dulbecco 's modified eagle 's medium ( dmem ) , glutmax , penicillin / streptomycin , and horse serum were purchased from invitrogen ( carlsbad , ca ) and fbs from hyclone ( logan , ut ) . all other materials were purchased from sigma aldrich ( st . louis , mo ) . sprague - dawley rats ( 50100 g ) , obtained from charles river laboratories ( framingham , ma ) , were housed in the boston university school of medicine animal facility . animals were on a 12-h light / dark cycle and were allowed standard rodent diet and water ad libitum . all studies were conducted according to the institutional animal care and use committee ( iacuc ) at boston university . on the experimental day , rats were anesthetized with sodium pentobarbital ( 60 mg / kg i.p . ) and edl muscles were removed and tied to stainless steel clips as described previously ( 17 ) . muscles were preincubated for 20 min at 37c in oxygenated ( 95% o2/5% co2 ) krebs - henseleit solution containing 6 mmol / l glucose and then incubated in the absence or presence of rat recombinant il-6 ( 15120 ng / ml ) for the times indicated ( 10120 min ) . for palmitate oxidation measurements , muscles were incubated with or without il-6 ( 120 ng / ml ) in the presense of 0.2 mmol / l palmitate complexed to 2% bsa in media containing 0.2 ci / ml [ u - c ] palmitate . after 30 min , media was acidified with acetic acid ( 0.5 n ) and the co2 released was trapped on hyamine hydroxide  soaked filter paper . at the end of the incubation protocols , muscles were blotted , quick - frozen in liquid nitrogen , and stored at 80c until additional analyses were performed . male sprague - dawley rats weighing 100 g ( 5 g ) were used for experiments in which they were injected with il-6 ( 25 ng / g animal weight i.p . ) . sixty minutes after the injection , animals were anesthetized with sodium pentobarbital ( 60 mg / kg i.p . ) and whole gastrocnemius muscle was excised , frozen in liquid nitrogen , and stored at 80c until further analysis . the cells were placed in six - well plates and cultured in dmem containing 1% glutmax , 1% penicillin / streptomycin , and 10% fbs . when myoblasts reached 80% confluence , their differentiation to myotubes was induced by switching the media to dmem containing 2% horse serum , 1% glutmax , and 1% penicillin / streptomycin . ampk activity was assayed in frozen skeletal muscle homogenized in lysis buffer as described previously ( 18 ) . briefly , muscle lysate containing 200 g protein was immunoprecipitated with antibody specific to the 2 or 1 catalytic subunit of ampk and protein a / g agarose beads . beads were washed five times , and the activity of the immobilized enzyme was assayed based on the phosphorylation of sams  peptide ( 0.2 mmol / l ) by 0.2 mmol / l atp ( containing 2 ci [ -p ] atp ) in the presence and absence of 0.2 mmol / l amp . cultured cells were scraped on ice in cell lysis buffer ( cell signaling technology , beverly , ma ) ( plus 1 mmol / l phenylmethylsulfonyl fluoride ) and centrifuged ( 14,000 g for 15 min at 4c ) . protein concentrations of cell supernatants were determined with the bicinchoninic acid reagents ( pierce , rockford , il ) using bsa as the standard . fifty micrograms of protein lysate from skeletal muscle or c2c12 cells were run on a 415% gradient polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane . membranes were then stained with ponceau s ( 1% in 5% acetic acid ) to ensure even transfer and blocked in tris - buffered saline ( ph 7.5 ) containing 0.05% tween 20 ( tbst ) and 5% milk for 1 h at room temperature . blots were first incubated overnight in primary antibody diluted in tbst containing 5% bsa ( 1:1,000 for p - ampk , p - acc , p - gsk3 , t - gsk3 ; 1:2,000 for p - stat3 and pgc1 ; and 1:500 for p - creb and ucp3 ) and then in tbst containing 5% nonfat dry milk and the appropriate secondary antibody conjugated to horseradish peroxidase at a 1:5,000 dilution . after this , the membranes were subjected to an enhanced chemiluminescence solution , and multiple autoradiogaphs ( to ensure linearity ) were generated . concentrations of atp , adp , amp , and phosphocreatine were initially determined spectrophotometrically in neutralized perchloric acid filtrates as described previously ( 19 ) . camp content was determined with a commercially available enzyme - linked immunosorbent assay kit according to the instructions provided by the manufacturer ( alpco diagnostics ) . samples assayed for camp were digested in 5% trichloroacetic acid ( tca ) , and the supernatant was extracted five times in an equal volume of diethyl ether . the aqueous layer was dried using the speedvac sc110 , and pellets were stored at 80c until analysis . on the day of analysis , pellets were reconstituted in 250 l of deionized water . in studies in which both camp and adenine nucleotides were assayed , tissues were extracted with tca . lactate release into the incubation medium was determined spectrophotometrically as described previously ( 20 ) , and glycerol release was determined with a commercially available kit after concentration of the medium by lyophilization ( sigma ) . tissue glycogen content was determined using the phenol - sulfuric acid reaction ( 21 ) . statistical significance was determined by a one - way or two - way anova for nonrepeated measures , as appropriate . the bonferroni post hoc test was used in the event of a significant ( p < 0.05 ) ratio . on the experimental day , rats were anesthetized with sodium pentobarbital ( 60 mg / kg i.p . ) and edl muscles were removed and tied to stainless steel clips as described previously ( 17 ) . muscles were preincubated for 20 min at 37c in oxygenated ( 95% o2/5% co2 ) krebs - henseleit solution containing 6 mmol / l glucose and then incubated in the absence or presence of rat recombinant il-6 ( 15120 ng / ml ) for the times indicated ( 10120 min ) . for palmitate oxidation measurements , muscles were incubated with or without il-6 ( 120 ng / ml ) in the presense of 0.2 mmol / l palmitate complexed to 2% bsa in media containing 0.2 ci / ml [ u - c ] palmitate . after 30 min , media was acidified with acetic acid ( 0.5 n ) and the co2 released was trapped on hyamine hydroxide  soaked filter paper . at the end of the incubation protocols , muscles were blotted , quick - frozen in liquid nitrogen , and stored at 80c until additional analyses were performed . male sprague - dawley rats weighing 100 g ( 5 g ) were used for experiments in which they were injected with il-6 ( 25 ng / g animal weight i.p . ) . sixty minutes after the injection , animals were anesthetized with sodium pentobarbital ( 60 mg / kg i.p . ) and whole gastrocnemius muscle was excised , frozen in liquid nitrogen , and stored at 80c until further analysis . the cells were placed in six - well plates and cultured in dmem containing 1% glutmax , 1% penicillin / streptomycin , and 10% fbs . when myoblasts reached 80% confluence , their differentiation to myotubes was induced by switching the media to dmem containing 2% horse serum , 1% glutmax , and 1% penicillin / streptomycin . ampk activity was assayed in frozen skeletal muscle homogenized in lysis buffer as described previously ( 18 ) . briefly , muscle lysate containing 200 g protein was immunoprecipitated with antibody specific to the 2 or 1 catalytic subunit of ampk and protein a / g agarose beads . beads were washed five times , and the activity of the immobilized enzyme was assayed based on the phosphorylation of sams  peptide ( 0.2 mmol / l ) by 0.2 mmol / l atp ( containing 2 ci [ -p ] atp ) in the presence and absence of 0.2 mmol / l amp . cultured cells were scraped on ice in cell lysis buffer ( cell signaling technology , beverly , ma ) ( plus 1 mmol / l phenylmethylsulfonyl fluoride ) and centrifuged ( 14,000 g for 15 min at 4c ) . protein concentrations of cell supernatants were determined with the bicinchoninic acid reagents ( pierce , rockford , il ) using bsa as the standard . fifty micrograms of protein lysate from skeletal muscle or c2c12 cells were run on a 415% gradient polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane . membranes were then stained with ponceau s ( 1% in 5% acetic acid ) to ensure even transfer and blocked in tris - buffered saline ( ph 7.5 ) containing 0.05% tween 20 ( tbst ) and 5% milk for 1 h at room temperature . blots were first incubated overnight in primary antibody diluted in tbst containing 5% bsa ( 1:1,000 for p - ampk , p - acc , p - gsk3 , t - gsk3 ; 1:2,000 for p - stat3 and pgc1 ; and 1:500 for p - creb and ucp3 ) and then in tbst containing 5% nonfat dry milk and the appropriate secondary antibody conjugated to horseradish peroxidase at a 1:5,000 dilution . after this , the membranes were subjected to an enhanced chemiluminescence solution , and multiple autoradiogaphs ( to ensure linearity ) were generated . concentrations of atp , adp , amp , and phosphocreatine were initially determined spectrophotometrically in neutralized perchloric acid filtrates as described previously ( 19 ) . camp content was determined with a commercially available enzyme - linked immunosorbent assay kit according to the instructions provided by the manufacturer ( alpco diagnostics ) . samples assayed for camp were digested in 5% trichloroacetic acid ( tca ) , and the supernatant was extracted five times in an equal volume of diethyl ether . the aqueous layer was dried using the speedvac sc110 , and pellets were stored at 80c until analysis . on the day of analysis , pellets were reconstituted in 250 l of deionized water . in studies in which both camp and adenine nucleotides were assayed , tissues were extracted with tca . lactate release into the incubation medium was determined spectrophotometrically as described previously ( 20 ) , and glycerol release was determined with a commercially available kit after concentration of the medium by lyophilization ( sigma ) . tissue glycogen content was determined using the phenol - sulfuric acid reaction ( 21 ) . statistical significance was determined by a one - way or two - way anova for nonrepeated measures , as appropriate . the bonferroni post hoc test was used in the event of a significant ( p < 0.05 ) ratio . the time course and dose response of ampk activation by il-6 are presented in fig . 1a and b. incubation of edl muscle with il-6 ( 120 ng / ml ) has been shown previously to induce a twofold increase in the phosphorylation of the  subunit of ampk at thr ( an index of its activity ) after 15 min , with values returning to baseline by 60 min ( 15 ) . 1a , an almost identical pattern was observed when the activity of the immunoprecipitated 2 isoform of ampk was measured . in contrast , no change in the activity of the 1 isoform of ampk was observed ( data not shown ) . activation of the 2 ampk occurred at il-6 concentrations as low as 15 ng / ml ; however , it was somewhat less than that observed at 120 ng / ml ( fig . incubation with il-6 ( 120 ng / ml ) for 15 min increased the phosphorylation of ampk ( thr 172 ) and of its downstream target acc ( ser ) ( fig . 1c ) in keeping with the changes in ampk 2 activity . thereafter , p - ampk and p - acc returned to baseline values after 60 min and remained so for up to 4 h , the maximum length of incubation tested ( data not shown ) . il-6 also increased the phosphorylation of stat3 ( tyr 705 ) , a known event in the activation of the jak / stat signaling cascade ( 22 ) ( fig . finally , although the increase in ampk activity was transient , it was followed by two- and fourfold increases in ucp3 protein abundance after 1 and 2 h , respectively ( fig . was observed ; however , it only achieved statistical significance after 2 h ( fig . incubation with il-6 increases 2 ampk activity , protein phosphorylation , and ucp3 and pgc1 protein abundance in incubated edl muscle . a : 2 ampk activity ( bar graph ) and amp : atp ratio ( line graph ) were assayed in rat edl muscles incubated with il-6 ( 120 ng / ml ) for 1560 min . b : dose - curve of 2 ampk activity in response to il-6 ( 15120 ng / ml ) for 30 min . c : immunoblots of p - acc ( ser ) , p - stat3 ( tyr ) , p - ampk ( thr ) , p - creb ( ser ) , and -actin in edl muscles incubated with il-6 ( 120 ng / ml ) for 15 min . d : immunoblots of pgc1 , ucp3 , and -actin in edl incubated with il-6 ( 120 ng / ml ) for 30120 min . e : densitometric analysis of immunoblots of ucp3 and pgc1 protein shown in d. results are means  se , n = 410 . edl muscles . * immunoblots shown are representative of four samples . as a first step in studying the mechanism by which il-6 activates ampk , we examined its effect on cellular energy state . control muscles gave similar values to those reported previously ( 23 ) ; however , incubation with il-6 ( 120 ng / ml ) led to decreases in the concentrations of creatine phosphate and atp , no significant changes in adp , and an increase in amp levels ( table 1 ) . this resulted in increases in the amp : atp ratio that paralleled those of ampk activity , with both of them being maximal at 15 min and returning to baseline by 60 min ( fig . il-6 alters intracellular concentrations of adenine nucleotides and creatine phosphate data are means  se ( n = 5 - 8 ) . rat edl muscles were incubated with il-6 ( 120 ng / ml ) for 1060 min . because no significant differences in the nucleotide content were observed in the control muscles at the different time points , values for control muscles at all time points were pooled for statistical purposes . ( 8) have reported that the injection of adrenaline activates ampk and increases the amp : atp ratio in rat adipose tissue and that these changes are inhibited by the -adrenergic antagonist propranolol . here we found that propranolol , at a concentration of 10 mol / l , also inhibited il-6induced ampk activation , raising the possibility that il-6 caused -adrenergic stimulation ( fig . in keeping with this notion , 15-min incubation with il-6 ( 120 ng / ml ) induced an increase in both camp levels and the phosphorylation of its downstream target p - creb at ser ( figs . in addition , propranolol prevented the increases in the amp : atp ratio caused by il-6 ( fig . 2a ) . as shown in fig . 1a , the alterations in the amp : atp ratio ( fig . 2a , line - graph ) coincided with changes in 2 ampk activity ( fig . 2a , bar graph ) . finally , consistent with these results , activation of ampk by il-6 il-6induced increases in 2 ampk activity , camp levels , and amp : atp ratio are abrogated by propanolol ( propr ) . a : activity of 2 ampk ( bar graph ) and amp : atp ratio ( line graph ) in edl pretreated or not pretreated for 30 min with the -adrenergic antagonist propranolol ( 10 mol / l ) and then with or without il-6 ( 120 ng / ml ) for 15 min . b : camp levels in edl muscles incubated with il-6 ( 120 ng / ml ) for 15 or 30 min . mol / l ) for 30 min and then with or without il-6 ( 120 ng / ml ) for 15 min . results are means  se ( n = 47 ) , * p < 0.05 versus control , no propanalol , p < 0.05 versus il-6 no propanalol , p < 0.05 versus 15-min time point . activity of 2 ampk in edl pretreated (  ) or not pretreated (  ) with dideoxyadenosine ( 100 mol / l ) for 30 min and then with or without il-6 ( 120 ng / ml ) for 15 min . results are means  se ( n = 46 ) , * p < 0.05 versus control , no inhibitor , p < 0.05 versus il-6 no inhibitor . treatment of c2c12 cells with il-6 increased camp , p - acc , p - ampk , p - stat3 , and p - creb abundance , as did isoproterenol ( fig . 4a and c ) , suggesting both that the effects of il-6 on the edl are not unique to the incubated muscle and that they can be mimicked by isoproterenol , a known -adrenergic agonist . in keeping with the former conclusion , il-6 injected intraperitoneally for 60 min also led to increases in the abundance of p - acc , p - ampk , p - stat3 , and p - creb and in the amp : atp ratio in the gastrocnemius muscle , just as it did in the incubated muscle ( fig . . immunoblots of p - acc ( ser ) , p - stat3 ( tyr ) , p - ampk ( thr ) , p - creb ( ser ) , and -actin were performed on ( a ) lysates of c2c12 myotubes treated with il-6 ( 50 ng / ml ) or isoproterenol ( iso ) ( 10 mol / l ) for 30 min and ( b ) homogenates of gastrocnemius muscle of rats injected intraperitoneally with saline ( veh ) or 2.5 g of il-6 1 h before being killed . c : camp levels assayed in extracts of c2c12 myotubes treated for 15 min with il-6 ( 50 ng / ml ) . d : adenine nucleotide and camp levels in muscles studied in b. results are means  se , n = 34 . il-6 has been reported to increase whole - body lipolysis ( 5 ) and decrease glycogen content in primary hepatocytes ( 24 ) , as do catecholamines , glucagon , and other agents that increase camp . 5a , incubation of the edl with il-6 ( 120 ng / ml ) for 120 min diminished muscle glycogen by 30% . in keeping with this , il-6 treatment decreased the phosphorylation of gsk3 suggesting that it inhibited glycogen synthase activity ( fig . the decrease in gsk3 phosphorylation was not affected by propranolol , indicating that it is independent of ampk activation . il-6 also caused a 1.5-fold increase in lactate release into the media and increased the release of glycerol , suggesting that glycolysis and lipolysis were also stimulated by this treatment ( fig . finally , in agreement with previous studies in cultured myotubes and soleus muscle ( 25 ) , incubation of the edl for 30 min with il-6 caused a fivefold increase in palmitate oxidation ( fig . incubated edl muscles were treated with (  ) or without (  ) il-6 ( 120 ng / ml ) for times indicated . b : representative immunoblot blots and densitometric analysis of p - gsk3 and t - gsk3. c and d : release into the medium of lactate and glycerol . e : fatty acid oxidation in edl incubated with il-6 ( 120 ng / ml ) in the presence of 0.2 ci / ml c - palmitate and 0.2 mmol / l palmitate bound to fatty acid palmitate oxidation was quantified by measuring the concentration of co2 released into the medium over the next 30 min ( see research design and methods ) . * p < 0.05 and p < 0.01 versus control , p < 0.05 versus 60 min il-6 ,  p < 0.05 versus 30 min il-6 . the time course and dose response of ampk activation by il-6 are presented in fig . 1a and b. incubation of edl muscle with il-6 ( 120 ng / ml ) has been shown previously to induce a twofold increase in the phosphorylation of the  subunit of ampk at thr ( an index of its activity ) after 15 min , with values returning to baseline by 60 min ( 15 ) . 1a , an almost identical pattern was observed when the activity of the immunoprecipitated 2 isoform of ampk was measured . in contrast , no change in the activity of the 1 isoform of ampk was observed ( data not shown ) . activation of the 2 ampk occurred at il-6 concentrations as low as 15 ng / ml ; however , it was somewhat less than that observed at 120 ng / ml ( fig . incubation with il-6 ( 120 ng / ml ) for 15 min increased the phosphorylation of ampk ( thr 172 ) and of its downstream target acc ( ser ) ( fig . 1c ) in keeping with the changes in ampk 2 activity . thereafter , p - ampk and p - acc returned to baseline values after 60 min and remained so for up to 4 h , the maximum length of incubation tested ( data not shown ) . il-6 also increased the phosphorylation of stat3 ( tyr 705 ) , a known event in the activation of the jak / stat signaling cascade ( 22 ) ( fig . finally , although the increase in ampk activity was transient , it was followed by two- and fourfold increases in ucp3 protein abundance after 1 and 2 h , respectively ( fig . was observed ; however , it only achieved statistical significance after 2 h ( fig . incubation with il-6 increases 2 ampk activity , protein phosphorylation , and ucp3 and pgc1 protein abundance in incubated edl muscle . a : 2 ampk activity ( bar graph ) and amp : atp ratio ( line graph ) were assayed in rat edl muscles incubated with il-6 ( 120 ng / ml ) for 1560 min . b : dose - curve of 2 ampk activity in response to il-6 ( 15120 ng / ml ) for 30 min . c : immunoblots of p - acc ( ser ) , p - stat3 ( tyr ) , p - ampk ( thr ) , p - creb ( ser ) , and -actin in edl muscles incubated with il-6 ( 120 ng / ml ) for 15 min . d : immunoblots of pgc1 , ucp3 , and -actin in edl incubated with il-6 ( 120 ng / ml ) for 30120 min . e : densitometric analysis of immunoblots of ucp3 and pgc1 protein shown in d. results are means  se , n = 410 . edl muscles . * as a first step in studying the mechanism by which il-6 activates ampk , we examined its effect on cellular energy state . control muscles gave similar values to those reported previously ( 23 ) ; however , incubation with il-6 ( 120 ng / ml ) led to decreases in the concentrations of creatine phosphate and atp , no significant changes in adp , and an increase in amp levels ( table 1 ) . this resulted in increases in the amp : atp ratio that paralleled those of ampk activity , with both of them being maximal at 15 min and returning to baseline by 60 min ( fig . il-6 alters intracellular concentrations of adenine nucleotides and creatine phosphate data are means  se ( n = 5 - 8 ) . rat edl muscles were incubated with il-6 ( 120 ng / ml ) for 1060 min . because no significant differences in the nucleotide content were observed in the control muscles at the different time points , values for control muscles at all time points were pooled for statistical purposes . 8) have reported that the injection of adrenaline activates ampk and increases the amp : atp ratio in rat adipose tissue and that these changes are inhibited by the -adrenergic antagonist propranolol . here we found that propranolol , at a concentration of 10 mol / l , also inhibited il-6induced ampk activation , raising the possibility that il-6 caused -adrenergic stimulation ( fig . in keeping with this notion , 15-min incubation with il-6 ( 120 ng / ml ) induced an increase in both camp levels and the phosphorylation of its downstream target p - creb at ser ( figs . in addition , propranolol prevented the increases in the amp : atp ratio caused by il-6 ( fig . 2a ) . as shown in fig . 1a , the alterations in the amp : atp ratio ( fig . 2a , line - graph ) coincided with changes in 2 ampk activity ( fig . 2a , bar graph ) . finally , consistent with these results , activation of ampk by il-6 il-6induced increases in 2 ampk activity , camp levels , and amp : atp ratio are abrogated by propanolol ( propr ) . a : activity of 2 ampk ( bar graph ) and amp : atp ratio ( line graph ) in edl pretreated or not pretreated for 30 min with the -adrenergic antagonist propranolol ( 10 mol / l ) and then with or without il-6 ( 120 ng / ml ) for 15 min . b : camp levels in edl muscles incubated with il-6 ( 120 ng / ml ) for 15 or 30 min . mol / l ) for 30 min and then with or without il-6 ( 120 ng / ml ) for 15 min . n = 47 ) , * p < 0.05 versus control , no propanalol , p < 0.05 versus il-6 no propanalol , p < 0.05 versus 15-min time point . activity of 2 ampk in edl pretreated (  ) or not pretreated (  ) with dideoxyadenosine ( 100 mol / l ) for 30 min and then with or without il-6 ( 120 ng / ml ) for 15 min . results are means  se ( n = 46 ) , * p < 0.05 versus control , no inhibitor , p < 0.05 versus il-6 no inhibitor . treatment of c2c12 cells with il-6 increased camp , p - acc , p - ampk , p - stat3 , and p - creb abundance , as did isoproterenol ( fig . 4a and c ) , suggesting both that the effects of il-6 on the edl are not unique to the incubated muscle and that they can be mimicked by isoproterenol , a known -adrenergic agonist . in keeping with the former conclusion , il-6 injected intraperitoneally for 60 min also led to increases in the abundance of p - acc , p - ampk , p - stat3 , and p - creb and in the amp : atp ratio in the gastrocnemius muscle , just as it did in the incubated muscle ( fig . il-6 exerts similar effects in c2c12 myotubes and gastrocnemius muscle in vivo . immunoblots of p - acc ( ser ) , p - stat3 ( tyr ) , p - ampk ( thr ) , p - creb ( ser ) , and -actin were performed on ( a ) lysates of c2c12 myotubes treated with il-6 ( 50 ng / ml ) or isoproterenol ( iso ) ( 10 mol / l ) for 30 min and ( b ) homogenates of gastrocnemius muscle of rats injected intraperitoneally with saline ( veh ) or 2.5 g of il-6 1 h before being killed . c : camp levels assayed in extracts of c2c12 myotubes treated for 15 min with il-6 ( 50 ng / ml ) . d : adenine nucleotide and camp levels in muscles studied in b. results are means  se , n = 34 . * il-6 has been reported to increase whole - body lipolysis ( 5 ) and decrease glycogen content in primary hepatocytes ( 24 ) , as do catecholamines , glucagon , and other agents that increase camp . 5a , incubation of the edl with il-6 ( 120 ng / ml ) for 120 min diminished muscle glycogen by 30% . in keeping with this , il-6 treatment decreased the phosphorylation of gsk3 suggesting that it inhibited glycogen synthase activity ( fig . the decrease in gsk3 phosphorylation was not affected by propranolol , indicating that it is independent of ampk activation . il-6 also caused a 1.5-fold increase in lactate release into the media and increased the release of glycerol , suggesting that glycolysis and lipolysis were also stimulated by this treatment ( fig . finally , in agreement with previous studies in cultured myotubes and soleus muscle ( 25 ) , incubation of the edl for 30 min with il-6 caused a fivefold increase in palmitate oxidation ( fig . incubated edl muscles were treated with (  ) or without (  ) il-6 ( 120 ng / ml ) for times indicated . a : glycogen content . b : representative immunoblot blots and densitometric analysis of p - gsk3 and t - gsk3. c and d : release into the medium of lactate and glycerol . e : fatty acid oxidation in edl incubated with il-6 ( 120 ng / ml ) in the presence of 0.2 ci / ml c - palmitate and 0.2 mmol / l palmitate bound to fatty acid palmitate oxidation was quantified by measuring the concentration of co2 released into the medium over the next 30 min ( see research design and methods ) . * p < 0.05 and p < 0.01 versus control , p < 0.05 versus 60 min il-6 ,  p < 0.05 versus 30 min il-6 . il-6 acutely activates ampk in skeletal muscle both in vivo and in vitro ( 15,16,25,26 ) . the results of the present study indicate that it does so by altering events that increase intracellular camp and , secondarily , the amp : atp ratio . in support of this notion , il-6induced ampk activation in the incubated edl was accompanied by increases in camp , and this , as well as the increase in the amp : atp ratio , was completely inhibited by the -adrenergic antagonist propranolol . additionally , the adenyl cyclase inhibitor 25 dideoxyadenosine blocked the activation of ampk by il-6 . finally , il-6 caused similar changes in c2c12 myotubes and gastrocnemius muscle in vivo , indicating its effects were not unique to the incubated edl . how the il-6induced elevation of camp in turn increased the amp : atp ratio remains to be determined . another agent that increases camp in skeletal muscle , isoproterenol , also has been reported to increase ampk activity ( 27 ) , lipolysis , and glycogenolysis ( 28 ) ; however , its effect on energy state was not examined . on the other hand , investigations in 3t3-l1 adipocytes have demonstrated that increases in the amp : atp ratio and ampk activation occur when camp is increased by either forskolin or isoproterenol ( 11 ) and that they are dependent on the stimulation of lipolysis . ( 8) have suggested that a similar mechanism occurs in rat adipose tissue in vivo , in that exercise and adrenaline ( epinephrine)-induced increases in the amp : atp ratio and ampk activity were both inhibited by propranolol . recently , it has been reported that il-6 induces lipolysis in porcine adipocytes and that this effect appears to be dependent on the actions of il-6 on erk1/2 and its ability to directly phosphorylate hormone - sensitive lipase ( hsl ) ( 29 ) ; however , the role of ampk in this setting was not examined . whether the stimulation of lipolysis accounts for the il-6induced changes in energy state and ampk activity in the edl muscle as it does in adipose tissue remains to be determined . it has been clearly demonstrated that camp and protein kinase a ( pka ) activation increase the biosynthesis of il-6 in various cells ( 30 ) ; however , il-6induced increases in camp have not been previously reported . we speculate that an intermediary molecule could be protein kinase c- ( pkc ) , given that this enzyme mediates the activation of the camp producing adenyl cyclase isoform vii ( 31 ) and that il-6 induces its membrane translocation in hepatocytes ( 32 ) . also , pkc mediates the binding of the il-6 transmembrane receptor gp130 to stat3 ( 33,34 ) , and it has been implicated in il-6induced insulin signaling ( 34 ) . the effects of il-6 on the phosphorylation of creb ( ser ) have not been reported previously ; however , it has been noted that both ampk and pka phosphorylate creb at the same residue ( ser ) ( 35 ) . in addition , both ampk ( 36,37 ) and creb ( 38 ) appear to regulate the abundance of ucp3 and pgc1. whether ampk or pka is responsible for the il-6induced increases in ucp3 and pgc1 observed in the present study remains to be determined experimentally . it is noteworthy that these effects of il-6 are qualitatively similar to those produced by leptin and cntf ( 10,39 ) , both of which also induce a rapid and transient activation of ampk , increase the amp : atp ratio ( 9,10 ) , and activate the jak / stat signaling cascade ( 22 ) . recently , it has been reported that stat3 can localize in mitochondria and enhance oxidative phosphorylation ( 40 ) , suggesting that it could directly influence cellular atp levels . in previous reports , a concentration of il-6 ( 120 ng / ml ) well above that in plasma was used to induce ampk activation in incubated rat skeletal muscle ( 15,26,41 ) . in the present study , ampk activation was observed at il-6 concentrations as low as 15 ng / ml ; however , this is still significantly higher than the reported resting concentration of il-6 in the plasma of a rat ( 10300 pg / ml ) ( 42,43 ) . during sustained exercise , the muscle cell is most likely exposed to much higher concentrations of il-6 given that it is synthesizing il-6 and releasing it into the circulation at amounts sufficient to increase its plasma concentration dramatically . thus , in humans , il-6 release from muscle during exercise can result in plasma concentrations 50- to 100-fold higher than those at rest ( 44 ) and concentrations in the interstitial fluid of muscle 10- to 100-fold higher than those in plasma ( 45,46 ) . in one study , an interstitial fluid il-6 concentration of 1.2 ng / ml was observed in humans after low - grade repetitive exercise without a detectible change in plasma il-6 ( 46 ) . therefore , in the rat , if one assumes a low ( 10 pg / ml ) resting plasma il-6 level and an effect of exercise similar to that in humans , an interstitial fluid il-6 concentration between 10100 ng / ml would be achieved during intense exercise ; this , however , remains to be determined . it has been proposed that il-6 synthesized and released into the circulation by skeletal muscle during exercise is an endocrine signal from the muscle cell that is becoming fuel deficient ( 4 ) . in keeping with this notion , il-6 increases adipose tissue lipolysis and hepatic glycogenolysis ( during exercise ) in humans ( 5,47 ) and its release from muscle large increases in muscle interstitial fluid il-6 have been observed in humans after low - intensity exercise in the absence of a change in plasma il-6 . this , together with the results of the present study , suggest that il-6 could also act as an autocrine or paracrine factor that enhances lipolysis and glycogenolysis and activates ampk in the muscle cell during exercise ( fig . finally , the physiological relevance of il-6 in this setting is strongly suggested by the observation that both the ability to sustain exercise and activate ampk are impaired in il-6 mice ( 15,49,50 ) . paracrine functions of il-6 synthesized and released by muscle during exercise . during sustained intense exercise , il-6 is synthesized and released from skeletal muscle cells and increases muscle camp , amp : atp ratio , and ampk activity . this results in enhanced muscle fatty acid oxidaton , lipolysis , and glycogenolysis . in summary , the results indicate that il-6 , at concentrations hypothetically representative of those released during exercise , activates ampk in skeletal muscle by increasing camp production and , secondarily , the amp : atp ratio . they also indicate that this transient activation of ampk results in a more sustained increase in the protein expression of two ampk - mediated genes that act on mitochondria , pgc1 , and ucp3 . finally , they reveal that il-6 increases substrate availability within the muscle cell by increasing glycogenolysis and lipolysis . studies in il-6 mice have established that a lack of il-6 markedly inhibits ampk activation during exercise ( 15 ) , whether it also diminishes the increases in camp levels , and pgc1 and ucp3 expression caused by exercise , remains to be determined .
objectiveinterleukin-6 ( il-6 ) directly activates amp - activated protein kinase ( ampk ) in vivo and in vitro ; however , the mechanism by which it does so is unknown.research design and methodswe examined this question in skeletal muscle using an incubated rat extensor digitorum longus ( edl ) muscle preparation as a tool.resultsampk activation by il-6 coincided temporally with a nearly threefold increase in the amp : atp ratio in the edl . 
 the effects of il-6 on both ampk activity and energy state were inhibited by coincubation with propranolol , suggesting involvement of -adrenergic signaling . in keeping with this notion , 
 il-6 concurrently induced a transient increase in camp , and its ability to activate ampk was blocked by the adenyl cyclase inhibitor 25-dideoxyadenosine . 
 in addition , like other -adrenergic stimuli , il-6 increased glycogen breakdown and lipolysis in the edl . 
 similar effects of il-6 on ampk , energy state , and camp content were observed in c2c12 myotubes and gastrocnemius muscle in vivo , indicating that they were not unique to the incubated edl.conclusionsthese studies demonstrate that il-6 activates ampk in skeletal muscle by increasing the concentration of camp and , secondarily , the amp : atp ratio . 
 they also suggest that substantial increases in il-6 concentrations , such as those that can result from its synthesis by muscles during exercise , may play a role in the mobilization of fuel stores within skeletal muscle as an added means of restoring energy balance .