see detailed information on materials , virus construction , quantitative pcr , and adipocyte area measurement in the supplementary methods . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1  10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement , cycloheximide ( 100 g / ml ) was used to inhibit protein synthesis . for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid brl37344 was added to the medium at 50 ng / ml . the culture tubes were shaken at 400 rpm . the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . adipoq mice were created as previously described ( 5 ) on the 129/svev genetic background and had been back - crossed to c57bl/6 for five generations . the experiments using mouse models were carried out under the association for assessment and accreditation of laboratory animal care guidelines with approval of the animal care and use committee . for some animal studies , 1  10 pfu of purified adenovirus vectors was injected into the mouse tail vein ( 6 ) . 3t3-l1 and 3t3-l1car1 cells and induction of adipocyte differentiation were described in a previous publication ( 7 ) . op9 adipocyte differentiation was induced using knockout sr ( 8) . although both 3t3-l1 and op9 adipocytes secrete adiponectin into the medium , adiponectin in overnight cultured medium accumulated only to 0.1% of mouse serum adiponectin . therefore , co - culture or conditioned medium was used to increase adiponectin protein levels . for co - culture , adipocytes were cultured in the outside well of the bd biosciences ( franklin lakes , nj ) transwell co - culture system , and fao cells were grown on the protein and cytokine - permeable membrane of the insert well . fao cells were transduced with adenovirus encoding mouse adiponectin ( ad - acrp30 ) or green fluorescent protein ( gfp ) for 12 h in a regular 6-well plate , and then the inserts with transduced fao cells were washed and transferred to the co - culture plates with differentiated adipocytes in the bottom wells . this system allows increase of medium adiponectin levels ( 40% of mouse serum adiponectin ) without physical interaction between 3t3-l1 adipocytes and fao cells . in a separate setting , conditioned medium from ad - acrp30transduced fao cells was collected and used for increasing adiponectin levels in some of the studies , as indicated in figure legends . for hormone - stimulated lipolysis , bt2-camp ( 100 mol / l ) was added to culture medium and cells were incubated for 1 h. total protein was used for certain protein or protein phosphorylation measurements using western blots . for pka rii protein half - life measurement for the in vivo lipolysis assays , the selective 3-adrenergic agonist brl37344 was injected i.p . into mice at 5 g / g body wt ( 9 ) . blood samples were collected 0 , 10 , and 20 min after the injection . for the lipolysis assay of adipose explants , 20 mg of epididymal adipose tissue explants were cultured in dulbecco s modified eagle s medium with 0.5% fatty acid the medium samples were collected 30 , 60 , 120 , and 180 min after adding brl37344 . the level of free fatty acid ( ffa ) or glycerol was normalized to the weight of adipose explants . for primary adipocyte lipolysis assays , adipocytes were prepared from epididymal fat using collagenase with an optimized procedure ( 11,12 ) . briefly , epididymal fat tissue was minced and digested in a krebs - ringer bicarbonate ( krb ) buffer ( ph 7.4 ) supplemented with 3% fatty acid  free bsa fraction v , 0.5 mmol / l adenosine , and 1 mg / ml type i collagenase . after 40-min digestion and three washes , isolated adipocytes were resuspended in krb supplemented with 5 mmol / l glucose and 3% fatty acid free bsa . suspended adipocytes ( 6,000 cells ) were used for a lipolysis assay in 400 l krb buffer plus 3% fatty acid free bsa , 1 unit / ml adenosine deaminase , and 100 nmol / l ( -)-n-(2-phenyl - isopropyl)-adenosine ( basal only ) with or without brl37344 ( 50 ng / ml ) . after 1-h incubation at 37c , 200 l of infranatant was removed and stored at 20c . serum free glycerol and glycerol released from fat explants were measured using a free - glycerol kit ( sigma - aldrich co. , st . glycerol in the assay media of primary and 3t3-l1 adipocytes was measured using bioluminescence , which is very sensitive and especially well adapted when only small amounts of adipose tissue or cells are available ( 13,14 ) . statistical analysis was performed using the student t test or anova , followed by contrast test . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26  0.02 vs. 0.42  0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet  induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet  induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp  treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . adipocytes were treated with or without bt2-camp ( 100 mol / l ) for 1 h. glycerol levels in medium were measured . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp  transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 nmol / l ) for 1 h. glycerol in medium was measured . hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl  specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . we postulated that adiponectin might increase hsl ser565 phosphorylation via ampk to inhibit hsl . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . as expected , wortmannin treatment attenuated the inhibitory effects of insulin on glycerol release 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna  treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet  induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means  sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . 5a ) . furthermore , knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna  treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome  mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig . similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . to study the effects of adiponectin on lipid metabolism in adipocytes , adipoq mice were used . consistent with previous studies ( 5,15 ) , there were no significant differences of body weights between adipoq and wild - type ( wt ) littermate controls when the mice were fed normal chow ( data not shown ) . however , body fat percentage of adipoq mice was significantly lower than that of wt mice ( fig . epididymal fat mass was also significantly smaller in adipoq male mice ( 0.26  0.02 vs. 0.42  0.04 g ; vs. wt , p < 0.05 , n = 10 ) . adipocyte cross - sectional areas of fixed epididymal fat of adipoq mice were 20% smaller than those of wt control mice ( supplementary fig . when mice were fed a 60% ( calorie ) high - fat diet for 8 weeks , adipoq mice gained remarkably less body weight and adipose tissue mass and had smaller adipocyte area ( fig . similar to previous studies ( 5,16 ) , high - fat diet induced more severe insulin resistance in adipoq mice ( data not shown ) . therefore , these studies clearly show that adipoq mice have less fat mass and are resistant to high - fat diet  induced obesity . similar phenotypes have been reported by a group using an independent line of adipoq mice ( 17 ) . adiponectin knockout mice have less body fat and are resistant to high - fat diet  induced obesity . body composition of chow - fed 2-month - old male adipoq and wt mice was scanned using echomri ( a ) . then the mice were fed a high - fat diet ( 60% of calories from fat ) . body fat was measured after 8 weeks of high - fat feeding ( c ) . our study shows that neither adiponectin gene knockout nor overexpression has any significant effect on gene expression of the key enzymes of triglyceride synthesis in epididymal fat ( supplementary fig . 2 ) , which suggests that adiponectin may regulate lipid metabolism in adipocytes through a pathway other than de novo lipogenesis . previous animal studies have clearly demonstrated that adiponectin reduces circulating ffas ( 16,1820 ) . although serum ffa levels at the fed state were similar between chow - fed adipoq and wt control mice , prolonged fasting - induced increase of ffa was significantly higher in adipoq mice ( supplementary fig . in addition , high - fat diet further increased fasting blood ffa in adipoq mice ( supplementary fig . these results imply that adiponectin might suppress ffa release and prompted us to study the effects of adiponectin on lipolysis in adipocytes . initially , we used the selective 3-adrenergic receptor agonist brl37344 and adipoq mice to carry out an in vivo lipolysis study , in which increases of serum free glycerol were measured to represent hormone - stimulated lipolysis . as expected , brl37344 treatment robustly increased serum free glycerol and ffa levels in all mice . the magnitudes of increase of serum glycerol in adipoq mice were significantly higher than those in wt mice 10 or 20 min after brl37344 injection ( p < 0.05 , fig . second , to avoid influence from other systems or hormones that may be rapidly altered by brl37344 administration , in vitro lipolysis assays were conducted using epididymal adipose explants . results showed that there were no significant differences in glycerol release between adipose explants from adipoq and wt control in a basal condition ( fig . consistent with the in vivo lipolysis study , brl37344-stimulated glycerol releases were remarkably higher in adipose explants from adipoq mice compared with wt control mice ( p < 0.01 , fig . 2b ) . in the third study , lipolysis was assayed in primary mature adipocytes from adipoq and wt control mice . as shown in fig . 2c , basal medium free glycerol levels were similar between adipocytes from wt control and adipoq mice . however , in bt2-camp  treated cells , free glycerol levels in the medium of adipoq adipocytes were twofold higher than those of adipocytes from wt mice ( fig . 2c ) . to further verify the effects of adiponectin on lipolysis , mouse embryonic fibroblast - derived adipocytes from adipoq mice were treated with or without adiponectin using a transwell co - culture system . results showed that camp - stimulated release of glycerol from adiponectin - treated adipocytes was significantly less than from control adipocytes ( supplementary fig . a : 3-adrenergic receptor agonist brl37344 was injected i.p . into 8-week - old male mice , which had been fasted overnight . n = 6 , * p < 0.05 vs. wt at the same time point . b : epididymal adipose explants were incubated in dulbecco s modified eagle s medium with or without brl37344 stimulation . n = 8 , * p < 0.05 vs. wt adipose explants with brl37344 stimulation . c : primary adipocytes were prepared from epididymal fat of adipoq or wt control mice . however , there was no significant difference in serum glucagon and insulin concentrations between chow - fed adipoq and wt mice ( data not shown ) , which prompted us to look at the expression and activation state of the main lipases in wat of adipoq mice . hsl and adipose triglyceride lipase ( atgl ) are two major lipases in adipocytes ( 21,22 ) . we examined the expression and activation status of these lipolytic proteins in epididymal adipose tissues from mice with adiponectin gene deletion or overexpression and from cultured adipocytes . for some animal studies , ad - acrp30 was used to reconstitute serum adiponectin in adipoq mice or to elevate serum adiponectin in c57bl/6 mice . as we previously reported ( 18 ) , 3 days after virus injection , total serum adiponectin protein levels were increased 12-fold in c57bl/6 mice , whereas fasting ffa levels were significantly reduced ( supplementary fig . serum multimeric adiponectin levels were proportionally elevated , and there were no significant changes in insulin sensitivity , as measured by an insulin challenge test ( data not shown ) ( 18 ) . as shown in fig . 3a , there were no significant differences of total protein levels of hsl , atgl , or cgi-58 in epididymal fat from adipoq , adiponectin - overexpressing , or adiponectin - reconstituted mice compared with their corresponding controls . in addition , adiponectin treatment did not alter protein levels of hsl , atgl , or cgi-58 in 3t3-l1 adipocytes ( fig . these results suggest that adiponectin - inhibited lipolysis is not through altering hsl , atgl , or cgi-58 protein levels in adipocytes . a : protein samples were prepared from epididymal fat of mice with indicated genotypes or treatment . relative protein levels of hsl , atgl , and cgi-58 , and serine phosphorylation levels of hsl were measured by western blot using specific antibodies . for adiponectin overexpression or reconstitution , ad - acrp30 or ad - gfp adenovirus b : mature 3t3-l1 adipocytes were co - cultured overnight with fao cells transduced with ad - acrp30 or ad - gfp . the insert wells with fao cells were removed , and adipocytes were treated with bt2-camp for 1 h. n = 6 . c : differentiated 3t3-l1 adipocytes were treated with cc ( 10 mol / l ) and co - cultured with ad - acrp30 or ad - gfp  transduced fao cells overnight . d : perilipin a protein levels in epididymal fat from adipoq and wt mice were measured using western blotting . e : for measuring pka - phosphorylated perilipin a , perilipin a proteins were immunoprecipitated and then probed with an antibody against phospho - pka substrate . f : after overnight co - culture with adiponectin secreting fao cells and wortmannin ( 200 nmol / l ) , 3t3-l1 adipocytes were stimulated with bt2-camp and insulin ( 25 hsl is the first lipase that was found to respond to lipolytic hormones and hydrolyze triglycerides in adipocytes . pka plays an essential role in mediating hormone - stimulated hsl activation and lipolysis in adipocytes . however , only ser660 phosphorylation activates hsl , whereas ser563 phosphorylation does not correlate with hsl activation ( 24 ) . by using phospho - hsl  specific antibodies , our study showed that the levels of hsl ser660 phosphorylation in epididymal fat from adipoq mice were strikingly elevated compared with those in wt control mice ( p < 0.001 , fig . 3a , bottom quantified graphs ) . in contrast , phosphorylation of hsl ser660 was significantly suppressed in epididymal fat of the mice with adenovirus - mediated adiponectin overexpression and adiponectin reconstitution ( p < 0.05 , fig . 3a ) , whereas a significantly reduced brl - stimulated glycerol release was observed in epididymal adipose explants of mice with adiponectin overexpression ( supplementary fig . 5a ) . in addition , significantly increased hsl ser660 phosphorylation was observed in the epididymal fat of adipoq mice fed a high - fat diet ( supplementary fig . these data indicate that hsl activity in wat is increased in adipoq mice and suppressed in mice with elevated adiponectin . by using 3t3-l1 adipocytes and co - culture system , our study further revealed that overnight adiponectin treatment significantly decreased both basal and camp - stimulated hsl ser660 phosphorylation ( p < 0.05 , fig . 3b ) . together , these results demonstrate that adiponectin suppresses hsl activation by reducing ser660 phosphorylation . it has been reported that ser565 in hsl can be phosphorylated by amp - activated protein kinase ( ampk ) , resulting in deactivation of hsl ( 21,25 ) . to our surprise , hsl ser565 phosphorylation levels neither decreased in adipoq mice nor increased in adiponectin - overexpressing or -reconstituted mice compared with their controls ( fig . these results exclude the possibility that adiponectin inhibits hsl activation by increasing hsl ser565 phosphorylation . to further determine the role of ampk in adiponectin - suppressed lipolysis , selective ampk inhibitor compound c ( cc ) was used . however , cc treatment did not abolish the inhibitory effects of adiponectin on glycerol release ( fig . similar results were obtained in 3t3-l1car1 adipocytes transduced with adenovirus encoding dominant negative ampk ( data did shown ) . these results do not support any key role of ampk in adiponectin - suppressed lipolysis . however , our study does not completely exclude the involvement of ampk in adiponectin - suppressed lipolysis . apparently , the underlying mechanisms through which ampk modulates lipolysis are far more complicated than a single serine site phosphorylation in hsl . studies have shown that pka or lipolysis itself induces ampk activation in adipocytes ( 26,27 ) . furthermore , a recent study reported that ampk not only suppresses but also increases lipolysis and that the effects are dependent on duration of ampk activation ( 28 ) . perilipin a is a lipid droplet - binding protein that plays an important role in lipolysis in adipocytes ( 29,30 ) . upon lipolytic hormone stimulation , pka - phosphorylated perilipin a recruits hsl to the lipid droplet surface ( 31 ) . our results showed that perilipin a protein levels in epididymal fat of adipoq mice were similar to those of wt control mice ( fig . these results suggest that pka - mediated perilipin a phosphorylation may be not responsible for adiponectin - inhibited lipolysis in adipocytes . adiponectin sensitizes insulin and insulin inhibits lipolysis ( 32 ) . this raises the question whether adiponectin inhibits lipolysis through insulin and whether there is a synergistic effect . we used the insulin signaling blocker wortmannin , which abolishes the inhibitory effects of insulin on lipolysis in adipocytes ( 32 ) . 3f ) . in addition , adipocytes treated with both adiponectin and insulin did not exhibit a synergistic reduction in glycerol release . adipor1 is ubiquitously expressed and is the main adiponectin receptor in white adipocytes ( 33 ) . to verify whether adiponectin inhibits hsl activation through the adiponectin receptor , adipor1 or sirna oligo against adipor1 was expressed using adenovirus vectors in 3t3-l1car1 adipocytes , which express adenovirus receptor to facilitate adenovirus - mediated gene transduction in mature adipocytes ( 35 ) . phosphorylation levels of hsl ser660 at both basal level and after camp stimulation were significantly reduced in ad - adipor1treated cells but were remarkably increased in adipor1 sirna  treated adipocytes ( supplementary fig . the coordinate changes of hsl ser660 phosphorylation in adipoq mice and adiponectin - treated adipocytes suggest that adiponectin might suppress hsl activation in adipocytes through reducing pka activity . with the use of an antibody that recognizes the consensus of phosphorylated pka substrate , our study showed that there are significant increases of pka substrate phosphorylation of proteins in epididymal fat from adipoq mice ( supplementary fig . 7a ) , which further supports the notion that adiponectin inhibits pka activation in adipocytes . however , adiponectin gene deletion has no significant impact on total camp levels in wat ( p > 0.05 , supplementary fig . pka type ii regulatory subunit ( rii ) has two distinct isoforms , rii and rii. rii is ubiquitously expressed , but rii is predominantly expressed in adipose , brain , and adrenal tissues . rii gene knockout mice are lean and resistant to high - fat diet  induced obesity ( 36 ) . we measured the gene expression levels of pka catalytic subunits and regulatory subunits ri and rii at both mrna and protein levels in epididymal fat from adipoq and adiponectin - restored mice . there were no significant changes of mrna levels of the two catalytic and four regulatory subunits of pka in adipoq mice ( data not shown ) . protein levels of two catalytic subunits , pka c and c , were also similar between adipoq and wt controls ( fig . 4a ) . rii but not rii protein levels were significantly elevated in epididymal fat of adipoq mice ( p < 0.05 , fig . 4a ) . in contrast , adiponectin reconstitution in adipoq mice remarkably reduced rii in the epididymal fat ( p < 0.05 , fig . a similar inhibitory effect of adiponectin on rii protein level was observed in 3t3-l1 adipocytes ( p < 0.05 , fig . 4b ) . these results led us to hypothesize that reduced rii mediates the inhibitory effect of adiponectin on hsl activation . a : total protein samples were prepared using epididymal fat from adipoq or ad - acrp30treated mice . b : fully differentiated 3t3-l1 adipocytes were co - cultured with ad - acrp30transduced fao cells , which secrete adiponectin . data are expressed as means  sem . to verify this hypothesis , we overexpressed rii in differentiated mature adipocytes . the levels of hsl ser660 phosphorylation were significantly increased in ad - pka riitreated 3t3-l1car1 adipocytes ( p < 0.05 , fig . overexpression of rii even slightly decreased hsl ser660 phosphorylation without statistical significance ( p > 0.05 , supplementary fig . most important , overexpression of rii abolished adiponectin - induced inhibition of hsl ser660 phosphorylation in 3t3-l1car1 adipocytes ( fig . knocking down rii in 3t3-l1car1 adipocytes significantly reduced both basal and camp - stimulated hsl ser660 phosphorylation ( fig . these results indicate that adiponectin suppresses hsl activation by reducing rii. pka rii overexpression attenuates the inhibitory effects of adiponectin on hsl ser660 phosphorylation . adenovirus vectors encoding gfp , pka rii ( a ) , or sirna against pka rii ( b ) were added to the medium . a : cells were co - cultured 12 h later with ad - acrp30transduced fao cells , which secrete adiponectin . b : some cells were treated with bt2-camp for 1 h. protein samples were prepared , and the phosphorylation levels of hsl ser660 and other proteins were detected using specific antibodies . * p < 0.05 vs. ad - lacz sirna  treated cells at basal state . the above studies show that adiponectin reduces rii protein levels without altering rii mrna level . therefore , we compared the protein half - life of rii between 3t3-l1 adipocytes treated with or without adiponectin . as shown in fig . rii protein levels were reduced by 30% after just 1 h of adiponectin treatment in the presence of cycloheximide ( fig . cycloheximide ( 100 g / ml ) was added to the medium , and the insert wells with fao cells were removed . b : 3t3-l1 adipocytes were treated with adiponectin - enriched conditioned medium from ad - acrp30transduced fao cells . c : proteasome inhibitor mg132 ( 25 mol / l ) was added to the medium 30 min before adiponectin treatment . d : mature 3t3-l1 adipocytes were co - cultured with adiponectin - expressing fao cells for 4 h. the pierce crosslink ip kit ( thermo scientific pierce protein research products , rockford , il ) was used for immunoprecipitation to eliminate nonspecific bands . * p < 0.05 vs. control cells at the same time point ( b ) or without mg132 treatment ( c ) . the ubiquitin - dependent proteasome is the most common system for protein degradation . to further investigate how adiponectin reduces rii , the proteasome - specific inhibitor mg-132 was added into medium 30 min before adiponectin treatment . 6c ) , which indicates that adiponectin enhances rii protein degradation through the ubiquitin - proteasome system . then , we measured ubiquitin levels of immunoprecipated rii protein from adiponectin - treated and control 3t3-l1 adipocytes . 6d shows , the levels of ubiquitinated rii protein were significantly elevated in adiponectin - treated 3t3-l1 adipocytes . together , these results indicate that adiponectin reduces rii protein levels by enhancing ubiquitin - proteasome  mediated protein degradation . it should be noted that overall protein ubiquitin levels were not clearly increased in adiponectin - treated cells ( supplementary fig similar effects of adiponectin on overall protein ubiquitination have also been observed in adiponectin - treated c2c12 myotubes ( data not shown ) . these results suggest that adiponectin - enhanced rii ubiquitination and protein degradation occur in a protein - specific manner . adipose tissue is the main energy depot in humans and some animals . during fasting and exercise , triglycerides are hydrolyzed to release ffas , which are oxidized by skeletal muscle , heart , and other tissues as fuel . with the discovery of adipocyte - derived hormones , the regulatory role of adipose - derived hormones on lipid metabolism has emerged as an important system in maintaining energy homeostasis . our current study provides strong evidence demonstrating that adiponectin suppresses hormone - stimulated lipolysis in mouse adipocytes . during the revision of this article , the same effects of adiponectin on lipolysis were reported in human adipocytes ( 37 ) . therefore , we propose that adiponectin plays an important role in maintaining energy homeostasis by suppressing lipolysis in wat . it has been reported that leptin , another adipose - derived hormone , directly increases lipolysis and enhances lipid depletion from adipocytes ( 38,39 ) . although it is uncertain whether leptin and adiponectin regulate lipolysis through the same pathway , these studies suggest that adiponectin and leptin exert opposite regulatory effects on lipolysis in adipocytes . in addition , the results from cultured adipocytes clearly indicate that adiponectin suppresses lipolysis through an autocrine loop . the discovery of the inhibitory effects of adiponectin on lipolysis will help us to understand how adiponectin improves circulating lipid profiles . however , it is premature to claim the importance of adiponectin - inhibited lipolysis in the development of obesity . there is a decrease of adiponectin gene expression in obesity ( 2 ) , which should attenuate the inhibitory effect of adiponectin on lipolysis , resulting in less lipid storage in fat . thus , obesity - associated low adiponectin expression may be beneficial in the context of obesity . in addition , despite lipolytic defects that have been observed in obese human and animal models ( 10,40 ) , the role of lipolytic defects in obesity is still uncertain . therefore , more studies are required to elucidate the role of adiponectin - suppressed lipolysis in the development of obesity and in energy metabolism under physiologic conditions . plasma ffa inversely associates with insulin sensitivity , especially in obese subjects ( 41,42 ) . our current study demonstrates that adiponectin inhibits lipolysis in wat , which should decrease the level of circulating ffa . therefore , we postulate that inhibition of lipolysis and reduction of ffa is a mechanism through which adiponectin enhances insulin sensitivity . on the other hand , the insulin - sensitizing effect of adiponectin however , our study demonstrated that insulin signaling is not responsible for adiponectin - inhibited lipolysis in adipocytes . furthermore , although high - fat feeding induces more severe insulin resistance in adipoq mice compared with wt control mice ( 16 ) , the inhibitory effects of adiponectin on lipolysis are still well preserved . therefore , these studies indicate that adiponectin inhibits hormone - stimulated adipocyte lipolysis through a mechanism(s ) other than insulin sensitizing . the essential role of pka in mediating hormone - stimulated lipolysis in adipocytes has been well established . it has been reported that rii gene knockout mice are lean and resistant to high - fat diet  induced obesity because of increased locomotor activity , energy expenditure , and lipolysis in wat ( 4345 ) . our study showed that adiponectin has no effect on rii expression and that rii overexpression even slightly reduces hsl ser660 phosphorylation in adipocytes . the current study does not provide any evidence to support the involvement of rii in adiponectin - inhibited lipolysis in adipocytes . in contrast , we found that the protein level of rii was elevated in adiponectin gene knockout mice . ectopic expression of rii abolished the inhibitory effects of adiponectin on hsl ser660 phosphorylation and lipolysis . conversely , knocking down rii reduced both basal and camp - stimulated hsl ser660 phosphorylation in adipocytes . these results led us to conclude that reduced rii mediates the inhibitory effects of adiponectin on lipolysis in adipocytes . it should be pointed out that by comparing the pka substrate phosphorylation profile and pka protein levels between adipoq and wt mice in different tissues ( data not shown ) , we found that the effects of adiponectin on pka subunit expression and pka activation occur in a tissue - specific manner . the mechanism by which rii mediates the inhibitory effect of adiponectin on lipolysis remains unknown . the large number of substrates and diverse downstream signaling pathways of pka , precise localization of pka to its specific substrate by a kinase - anchoring proteins ( akaps ) has been demonstrated to be an important mechanism to restrict pka action ( 46 ) . recent studies revealed that rii binds tightly with some akaps and plays an important role in compartmentalizing camp / pka signal transduction ( 4648 ) . we postulate that adiponectin suppresses rii expression , which may shift akaps / pka away from the compartment where hsl localizes , resulting in decreased hsl phosphorylation and activation . the absence of effect of adiponectin on perilipin a phosphorylation also supports this postulation . in conclusion , our study demonstrates that adiponectin inhibits lipolysis and directly regulates lipid metabolism in adipocytes . our data also suggest that adiponectin suppresses triglyceride hydrolysis by inhibiting pka - induced hsl activation .