dulbecco 's modified eagle medium ( dmem)/ham 's f-12 1:1 containing 1% bsa and supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , and 0.25 g / ml amphotericin b ( antibiotic  antimycotic , invitrogen ) . low - glucose dmem without leucine , lysine , and arginine ( sigma aldrich ) was supplemented with 0.105 g / l leucine , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 0.0159 g / l phenol red , and 100 nmol / l insulin ( dmem / k-/r- ) . isotopes of arginine ( cambridge laboratories u - c6 units  n4 arginine cnlm-539 or u - c6 arginine clm-2265 0.021 g / l ) and lysine ( cambridge laboratories u - c6 units - n2 lysine cnlm-291 or 4,4,5,5-d4 lysine cnlm-2640 0.0365 g / l ) were added to produce  heavy  and  medium  silac medium , respectively . endothelial cell growth medium ( ecgm ) is low - glucose dmem supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , 20 mmol / l hepes , 1% nonessential amino acids , 50 mmol / l 2-mercaptoethanol , 20% heat - inactivated fcs , 12 units / ml heparin , and 150 g / ml endothelial cell growth supplement ( sigma ) . endothelial cell silac medium is dmem / k-/r- supplemented with 20 mmol / l hepes , 1% nonessential amino acids , and 50 mmol / l 2-mercaptoethanol . preadipocyte growth medium is dmem / ham 's f-12 1:1 supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , and 10% fcs . animals were kept in a temperature - controlled room ( 22  1c ) , 80% relative humidity , on a 12-h light / dark cycle with free access to food and water . experiments were carried out with approval of garvan institute animal experimentation ethics committee , following guidelines issued by the national health and medical research council of australia . sheep anti - rat igg dynabeads ( invitrogen ) were precoated with rat anti - mouse monoclonal antibody to cd31 ( pecam-1 ) ( bd pharmingen ) according to the manufacturer 's instructions at a concentration of 1 g antibody per 1  10 dynabeads . dynabeads were stored in pbs with 0.1% bsa at 4c at 4  10 beads / ml . paired vat ( epididymal ) and sat ( inguinal ) depots were obtained from 6-week old c57bl/6j mice . fat pads were immediately transported in warm transfer medium to a tissue culture hood , where they were placed in fresh transfer medium and minced into 1 mm pieces . minced explants were rinsed in pbs and centrifuged at 250 g to remove cell debris and erythrocytes . adipose tissue explants ( 200 l ) were incubated in 1 ml silac medium . after 48 h of incubation at 37c in 5% co2 , fat pads were immediately transported in warm transfer medium to a tissue culture hood , where they were placed in fresh transfer medium and minced into 1 mm pieces . medium was removed and replaced with fresh transfer medium containing 1 mg / ml collagenase type i and incubated with gentle shaking at 37c for 1 h. adipose tissue to digest solution ratio was 4:1 . resulting material was filtered through 250 m mesh , and adipocytes and free oil were separated from stromovascular components by centrifugation at 250 g for 5 min at room temperature . the stromovascular pellet was resuspended , washed in pbs , and centrifuged at 250 g for 5 min at room temperature . the stromovascular pellet was resuspended and plated in 2% gelatin - coated 10 cm dishes in ecgm . cells were detached using 0.25% trypsin containing 3.42 mmol / l edta and trypsin neutralized by addition of hank 's balanced salt solution containing 5% fcs ( hbss - fcs ) . the cell pellet was resuspended in 0.5 ml hbss - fcs and incubated with 25 l of anti - cd31 ( anti - pecam-1 ) coated dynabeads for 15 min at 4c under constant rotation . the cell / bead suspension was brought to a total volume of 10 ml with hbss - fcs and mvecs selected by magnet . selected cells were resuspended in ecgm and transferred to 2% gelatin - coated culture dishes . after 35 days , cells were routinely passaged and plated at equal density in ecgm . nonselected cells in wash medium were centrifuged at 600 g for 5 min at room temperature . the resultant pellet was resuspended in preadipocyte growth medium and plated at equal density in 10 cm culture dishes . after 48 h , conditioned medium was collected and centrifuged at 2,000 g to pellet cell debris . conditioned media from paired visceral and subcutaneous depots were mixed in 1:1 ratio , and glycoprotein purification was performed as previously described ( 30 ) . briefly , mncl2 was added to conditioned media to a final concentration of 1 mmol / l . samples were incubated overnight at 4c under constant rotation with 50 l / ml of 50% slurry of concanavalin a sepharose beads ( ge healthcare ) . beads were washed extensively with cona binding buffer ( 0.5 mol / l nacl , 0.1 mol / l tris , 1 mmol / l mncl2 , and 1 mmol / l cacl2 , ph 7.4 ) . proteins were eluted with 0.3 mol / l methyl--d - mannopyranoside , 0.5 mol / l nacl , 0.1 mol / l tris , 10 mmol / l edta , and 10 mmol / l egta , ph 7.4 . peptide extraction and mass spectrometry were as previously described ( 30,32 ) using a waters ultima tandem mass spectrometer . swissprot database was used for protein identification , taxonomy restricted to mus musculus , mass spectrometry tolerance set at 0.5 da , and mass spectrometry / mass spectrometry tolerance set at 0.05 da . heavy ( u - c6 units - n4 arginine , u - c6 units - n2 lysine ) to light ( naturally occurring lysine and arginine ) ( h / l ) and  medium ( u - c6 arginine , 4,4,5,5-d4 lysine ) to light  ( m / l ) isotope ratios were used to determine relative abundance of proteins from visceral or subcutaneous samples , respectively . as visceral and subcutaneous samples are mixed , nonlabeled proteins form a  common denominator  that both isotopes are measured against ( fig . cilair workflow for comparing protein synthesis and secretion from two different samples . paired cell cultures or tissues are incubated in media containing different isotopes of arginine and lysine , which are subsequently incorporated into newly synthesized and secreted proteins . paired conditioned media samples are mixed in equal proportions , and proteins are subjected to mass spectrometry . incorporation of amino acid isotopes results in a shift in molecular weight and mass to charge ( m / z ) ratio . peak heights are measured , and their ratios are used to determine relative abundance of proteins from samples a and b. dulbecco 's modified eagle medium ( dmem)/ham 's f-12 1:1 containing 1% bsa and supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , and 0.25 g / ml amphotericin b ( antibiotic  antimycotic , invitrogen ) . low - glucose dmem without leucine , lysine , and arginine ( sigma aldrich ) was supplemented with 0.105 g / l leucine , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 0.0159 g / l phenol red , and 100 nmol / l insulin ( dmem / k-/r- ) . n4 arginine cnlm-539 or u - c6 arginine clm-2265 0.021 g / l ) and lysine ( cambridge laboratories u - c6 units - n2 lysine cnlm-291 or 4,4,5,5-d4 lysine cnlm-2640 0.0365 g / l ) were added to produce  heavy  and  medium  silac medium , respectively . endothelial cell growth medium ( ecgm ) is low - glucose dmem supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , 20 mmol / l hepes , 1% nonessential amino acids , 50 mmol / l 2-mercaptoethanol , 20% heat - inactivated fcs , 12 units / ml heparin , and 150 g / ml endothelial cell growth supplement ( sigma ) . endothelial cell silac medium is dmem / k-/r- supplemented with 20 mmol / l hepes , 1% nonessential amino acids , and 50 mmol / l 2-mercaptoethanol . preadipocyte growth medium is dmem / ham 's f-12 1:1 supplemented with 100 units / ml penicillin , 0.1 mg / ml streptomycin , and 10% fcs . animals were kept in a temperature - controlled room ( 22  1c ) , 80% relative humidity , on a 12-h light / dark cycle with free access to food and water . experiments were carried out with approval of garvan institute animal experimentation ethics committee , following guidelines issued by the national health and medical research council of australia . sheep anti - rat igg dynabeads ( invitrogen ) were precoated with rat anti - mouse monoclonal antibody to cd31 ( pecam-1 ) ( bd pharmingen ) according to the manufacturer 's instructions at a concentration of 1 g antibody per 1  10 dynabeads . dynabeads were stored in pbs with 0.1% bsa at 4c at 4  10 beads / ml . paired vat ( epididymal ) and sat ( inguinal ) depots were obtained from 6-week old c57bl/6j mice . fat pads were immediately transported in warm transfer medium to a tissue culture hood , where they were placed in fresh transfer medium and minced into 1 mm pieces . minced explants were rinsed in pbs and centrifuged at 250 g to remove cell debris and erythrocytes . adipose tissue explants ( 200 l ) were incubated in 1 ml silac medium . after 48 h of incubation at 37c in 5% co2 , fat pads were immediately transported in warm transfer medium to a tissue culture hood , where they were placed in fresh transfer medium and minced into 1 mm pieces . medium was removed and replaced with fresh transfer medium containing 1 mg / ml collagenase type i and incubated with gentle shaking at 37c for 1 h. adipose tissue to digest solution ratio was 4:1 . resulting material was filtered through 250 m mesh , and adipocytes and free oil were separated from stromovascular components by centrifugation at 250 g for 5 min at room temperature . the stromovascular pellet was resuspended , washed in pbs , and centrifuged at 250 g for 5 min at room temperature . the stromovascular pellet was resuspended and plated in 2% gelatin - coated 10 cm dishes in ecgm . cells were detached using 0.25% trypsin containing 3.42 mmol / l edta and trypsin neutralized by addition of hank 's balanced salt solution containing 5% fcs ( hbss - fcs ) . the cell pellet was resuspended in 0.5 ml hbss - fcs and incubated with 25 l of anti - cd31 ( anti - pecam-1 ) coated dynabeads for 15 min at 4c under constant rotation . the cell / bead suspension was brought to a total volume of 10 ml with hbss - fcs and mvecs selected by magnet . selected cells were resuspended in ecgm and transferred to 2% gelatin - coated culture dishes . after 35 days , cells were routinely passaged and plated at equal density in ecgm . nonselected cells in wash medium were centrifuged at 600 g for 5 min at room temperature . the resultant pellet was resuspended in preadipocyte growth medium and plated at equal density in 10 cm culture dishes . after 48 h , conditioned medium was collected and centrifuged at 2,000 g to pellet cell debris . conditioned media from paired visceral and subcutaneous depots were mixed in 1:1 ratio , and glycoprotein purification was performed as previously described ( 30 ) . briefly , mncl2 was added to conditioned media to a final concentration of 1 mmol / l . samples were incubated overnight at 4c under constant rotation with 50 l / ml of 50% slurry of concanavalin a sepharose beads ( ge healthcare ) . beads were washed extensively with cona binding buffer ( 0.5 mol / l nacl , 0.1 mol / l tris , 1 mmol / l mncl2 , and 1 mmol / l cacl2 , ph 7.4 ) . proteins were eluted with 0.3 mol / l methyl--d - mannopyranoside , 0.5 mol / l nacl , 0.1 mol / l tris , 10 mmol / l edta , and 10 mmol / l egta , ph 7.4 . peptide extraction and mass spectrometry were as previously described ( 30,32 ) using a waters ultima tandem mass spectrometer . swissprot database was used for protein identification , taxonomy restricted to mus musculus , mass spectrometry tolerance set at 0.5 da , and mass spectrometry / mass spectrometry tolerance set at 0.05 da . heavy ( u - c6 units - n4 arginine , u - c6 units - n2 lysine ) to light ( naturally occurring lysine and arginine ) ( h / l ) and  medium ( u - c6 arginine , 4,4,5,5-d4 lysine ) to light ( m / l ) isotope ratios were used to determine relative abundance of proteins from visceral or subcutaneous samples , respectively . as visceral and subcutaneous samples are mixed , nonlabeled proteins form a  common denominator  that both isotopes are measured against ( fig . cilair workflow for comparing protein synthesis and secretion from two different samples . paired cell cultures or tissues are incubated in media containing different isotopes of arginine and lysine , which are subsequently incorporated into newly synthesized and secreted proteins . paired conditioned media samples are mixed in equal proportions , and proteins are subjected to mass spectrometry . incorporation of amino acid isotopes results in a shift in molecular weight and mass to charge ( m / z ) ratio . peak heights are measured , and their ratios are used to determine relative abundance of proteins from samples a and b. adipose tissue explants from epididymal ( vat ) and inguinal ( sat ) depots were incubated for 48 h in the presence of  heavy  and  medium  isotopes of arginine and lysine , respectively . proteins were precipitated , resolved by sds - page , trypsin - digested , and subjected to quantitative mass spectrometry ( table 1 and supplementary data available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0483/dc1 ) . a total of 145 proteins were identified , of which 86 ( 60% ) were predicted to contain an nh2-terminal signal peptide using signalp . of these , 51 proteins ( 59% ) were present at > 2-fold higher abundance in conditioned medium from vat than sat , whereas only 18 proteins ( 21% ) were at > 2-fold higher abundance in conditioned medium from sat than vat . the frequency distribution of the visceral - to - subcutaneous ratio for detected proteins is shown in fig . the distribution for these background proteins with no signal peptide resembles a normal distribution , whereas the distribution for proteins with a signal peptide is skewed to the right . this indicates that release of true secretory proteins into conditioned medium is increased from vat relative to sat . the normal distribution of nonsecretory background proteins indicates a similar amount of tissue was present in vat and sat samples . comparative secretion of proteins identified from visceral and subcutaneous white adipose tissue explants visceral or subcutaneous wat explants were incubated in media containing different isotopes of arginine and lysine . conditioned media were mixed , subjected to lectin affinity chromatography , and subjected to quantitative mass spectrometry . peak height ratios of isotope to endogenous ( c ) amino acids and ratios between isotopes are shown . a : white adipose tissue ( wat ) explants secretory proteins are secreted in greater amounts from vat . b : preadipocyte secreted proteins are secreted at greater amounts from visceral - derived preadipocytes . c : endothelial cell secretory proteins are secreted at greater amounts from visceral - derived endothelial cells . wat explants , preadipocytes , and microvascular endothelial cells from visceral and subcutaneous wat depots were cultured in media containing two different isotopes of arginine and lysine . clustering analysis of detected proteins was performed using the database for annotation , visualization , and integrated discovery ( david ) ( 34,35 ) . analysis of proteins increased from vat revealed enrichment of proteins involved in endopeptidase inhibition , including members of the serpin family , and proteins involved in innate immune system regulation , including members of the complement pathway . adipose tissue is composed of adipocytes as well as preadipocytes , mvecs , and macrophages , collectively referred to as the stromovascular fraction . stromovascular cells may make a significant contribution to the secretory capacity of white adipose tissue , perhaps even exceeding that of adipocytes ( 36 ) . interplay between cells of the stromovascular fraction and adipocytes may also modulate the secretion profile of adipose tissue . we sought to determine the relative contribution of preadipocytes and mvecs to the whole adipose tissue secretome . in addition , we examined whether depot - specific differences in protein secretion observed from whole adipose tissue explants persisted in cells from the stromovascular fraction . visceral and subcutaneous preadipocytes were cultured in vitro for at least one week before incubation in medium containing isotopic amino acids . the frequency distribution of the visceral - to - subcutaneous ratio for detected proteins is shown in fig . this indicates that the release of secretory proteins into conditioned medium is increased from visceral relative to subcutaneous preadipocytes . cluster analysis of the proteins upregulated from visceral - derived preadipocytes showed an enrichment of extracellular matrix proteins . comparative secretion of proteins identified from visceral and subcutaneous adipose tissue  derived preadipocytes visceral or subcutaneous wat - derived preadipocytes were incubated in media containing different isotopes of arginine and lysine . conditioned media were mixed , subjected to lectin affinity chromatography , and subjected to quantitative mass spectrometry . peak height ratios of isotope to endogenous ( c ) amino acids and ratios between isotopes are shown . next , we examined mvecs isolated from paired vat and sat depots . to date the secretome of endothelial cells has not been determined . we isolated mvecs using cd31 magnetic bead separation and cultured these cells in vitro . conditioned media from cells incubated in isotopic amino acids were collected and analyzed as before ( table 3 and supplementary data ) . of these , 21 ( 62% ) were increased > 2-fold from visceral mvecs and only 5 were increased > 2-fold from subcutaneous mvecs . the frequency distribution of the visceral to subcutaneous ratio for detected proteins is shown in fig . 2c . once again , these data are skewed toward increased protein secretion from visceral mvecs ( fig . 2c ) . this is consistent with the enhanced secretory capacity of vat we have observed in both whole adipose tissue and isolated preadipocytes . the proteins identified include a number of proteins known to be secreted from endothelial cells , including thrombospondin-1 and thrombospondin-2 , and extracellular matrix proteins such as fibronectin and collagens . additionally , several proteins not previously known to be secreted from adipose tissue mvecs were identified . these include the most abundantly detected protein , adipocyte enhancer binding protein 1 ( aebp1 ) . this protein influences inflammation in macrophages via a direct interaction with pxr and ppar sites . cluster analysis showed proteins more abundantly secreted from visceral mvecs were involved in extracellular matrix formation , including collagen and fibronectins , and endopeptidase inhibition , including members of the serpin family . there were no defined clusters for the five secretory proteins more abundantly secreted from subcutaneous mvecs . comparative secretion of proteins identified from visceral and subcutaneous adipose tissue  derived endothelial cells visceral or subcutaneous wat - derived endothelial cells were incubated in media containing different isotopes of arginine and lysine . conditioned media were mixed , subjected to lectin affinity chromatography , and subjected to quantitative mass spectrometry . peak height ratios of isotope to endogenous ( c ) amino acids and ratios between isotopes are shown . when the secretomes of whole adipose tissue explants , preadipocytes , and mvecs were compared , eight common secreted proteins were identified ( fig . seven secreted proteins were shared between the whole adipose tissue and mvec secretomes , and four were shared between the whole adipose tissue and preadipocyte secretomes . these shared proteins included proteins involved in extracellular matrix formation , such as collagen and fibronectin , and other known adipocyte proteins known such as osteonectin . interestingly , these data suggest the majority of proteins in the whole adipose tissue secretome are secreted either by adipocytes themselves or by other cells in the stromovascular fraction such as macrophages and neurons . to interrogate this further , we compared the secretome of whole adipose tissue explants to the secretomes of differentiated 3t3-l1 adipocytes ( 30 ) and isolated rat adipocytes ( 27 ) . of the 86 proteins identified in the whole adipose tissue secretome , 26 were shared with the 3t3-l1 adipocyte secretome and 21 were shared with the isolated rat adipocyte secretome ( fig . it is possible that other cells types in the stromovascular fraction such as macrophages and neurons are the source of secreted proteins . to interrogate this further , we compared the secretome of whole adipose tissue explants to the secretome of human macrophages , and only three common proteins were identified ( 37 ) . ( a high - quality color representation of this figure is available in the online issue . ) venn diagram showing overlap of protein secretion from wat explants , 3t3l1 adipocytes , and isolated rat adipocytes ( from ref 27 ) . the 12 proteins detected in all three samples were adiponectin , adipsin , angiotensinogen ( serpina8 ) , cathepsin b , cathepsin d , collagen -1(iv ) , collagen -2(iv ) , complement c1s , haptoglobin , laminin subunit -2 , osteonectin , and thrombospondin-1 . ( a high - quality color representation of this figure is available in the online issue . ) first , the overall secretory capacity of vat is considerably higher than sat , and second , this enhanced secretory capacity of vat extends to cells of the stromovascular fraction  preadipocytes and mvecs . third , adipose tissue as a whole secretes more proteins than its individual component parts , suggesting the adipocyte is not the principal secretory component of adipose tissue . the secretome of adipose tissue is likely due to the complex interplay between its component cell types . to interrogate the adipose tissue secretome , we combined two techniques , cilair ( 29 ) and lac ( 30 ) . using this streamlined method excluded contaminating proteins from entrapped serum and intracellular contents from damaged cells . proteins already in the secretory pathway will remain unlabeled as will contaminants from serum and cell debris ( 38 ) . lac selectively enriches for proteins targeted for secretion , thus overcoming contamination from dead or damaged cells . this obviates the need for multiple washing steps to dilute these contaminating serum proteins , decreasing sample processing time and the negative impact of prolonged tissue handing . despite these measures , unlabeled proteins will remain in the medium . as conditioned media from visceral and subcutaneous samples are mixed , nonlabeled (  light  ) proteins form a  common denominator  that both isotopes are measured against . this eliminates any potential confounding from unlabeled contaminants differentially distributed between the two depots . the technique of lac cilair is a useful and valid tool for interrogation of the adipose tissue secretome , indicated by the detection of well - known adipokines including adiponectin , adipsin , and plasminogen activator inhibitor-1 ( pai-1 ) . of proteins secreted in > 2-fold abundance , 59% were from vat explants compared with 21% from sat explants . this upregulation of protein secretion from vat was consistent across cells of the stromovascular fraction with 68% of proteins secreted in > 2-fold abundance from vat preadipocytes compared with 0% from sat and 62% of proteins secreted in > 2-fold abundance from vat mvecs compared with 15% from sat ( table 4 ) . preadipocytes and mvecs were cultured in vitro for at least one week prior to application of labeled media so were no longer subject to paracrine influences from their depot of origin . the detection of a small number of proteins secreted in twofold greater abundance from subcutaneous depots provides evidence that our technique does not selectively detect enhanced protein secretion from the visceral depot . summary of numbers of proteins detected and relative abundance between visceral and subcutaneous adipose tissue samples cluster analysis of proteins secreted in greater abundance from vat revealed members of the acute phase response and innate immune system . the first is that vat is more pro - angiogenic than sat , as many members of the acute phase response , for example , haptoglobin ( 39 ) , and innate immune system ( 40 ) are involved in angiogenesis . the capacity for angiogenesis is a crucial requirement for adipose tissue expansion , and cells of the adipose tissue stromovascular fraction are able to differentiate into adipocytes and endothelial cells and vice versa ( 41 ) . anti - angiogenic agents have been proposed as novel therapeutic options in obesity as they induce weight loss in obese mouse models without adverse effect ( 42 ) . in support of this hypothesis , cluster analysis of the 14 proteins secreted more abundantly from visceral preadipocytes revealed proteins involved in extracellular matrix production . this , together with our finding that vat secretes pro - angiogenic factors , suggests that visceral preadipocytes are better adapted for rapid adipose tissue expansion than subcutaneous preadipocytes . this is consistent with recent data that vat , particularly in obesity , develops chronic low - grade inflammation with macrophage infiltration . this suggests that adipose tissue mvecs play a pivotal role in enticing circulating macrophages into adipose tissue . however , adipose tissue used for this study was derived from young , lean animals . in this setting , the upregulation of these cytokines is more likely targeted for angiogenesis and adipose tissue proliferation rather than a proinflammatory milieu . furthermore , recruitment of macrophages into adipose tissue is not always associated with inflammation as macrophages in adipose tissue from lean mice express il-10 , which protects adipocytes from insulin resistance ( 43 ) . this study represents the first survey of the endothelial cell secretome . in comparing mvecs from vat and sat , significant differences in protein secretion were demonstrated . among the endothelial secretome were thrombospondins 1 , 2 , and 3 , known to be secreted by endothelial cells , as well as a number of proteins not previously documented as endothelial secreted factors . surprisingly , many were known adipocyte secretory proteins , including growth arrest specific 6 , pai-1 , periostin , pentraxin - related protein 3 , and sparc . of particular interest was identification of aebp1 , a secreted protein proposed to act as a transcriptional repression factor through binding of the enhancer sequence of the ap2/fabp4 gene in adipocytes , as well as ppar and lxr sites in macrophages ( 44,45 ) preadipocytes deficient in aebp1 are hyperproliferative , with enhanced adipogenesis ( 46 ) . aebp1 can be detected in whole adipose tissue samples , but not when adipocytes are purified from adipose tissue ( 47 ) . given our discovery of aebp1 secretion from endothelial cells , we consider it likely that the source of aebp1 in adipose tissue is endothelial cells . in this study , we found that mvecs from vat secreted far more aebp1 than mvecs from sat . in this study , we interrogated the secretome of whole adipose tissue and subsequently cells of the stromovascular fraction , preadipocytes and mvecs , to determine which component of adipose tissue was principally responsible for adipokine secretion . a total of 115 proteins were identified from these three samples , of which 67 were unique to adipose tissue explants , 16 were unique to mvecs , and 7 were unique to preadipocytes . eight proteins were identified in all samples , including members of the collagen family , osteonectin , metalloproteinase inhibitor 1 , c1 esterase inhibitor , plasminogen activator inhibitor-1 , and cathepsin b. these data suggest adipocytes themselves or other cells in the stromovascular fraction not interrogated in this study , including macrophages and neurons , are the key secretory cells within adipose tissue . when the secretome of whole adipose tissue was compared with the secretomes of both differentiated 3t3-l1 adipocytes and primary rat adipocytes , only 26 ( 29% ) and 20 ( 22% ) common proteins , respectively , were identified . these data infer that adipocytes alone are not the principal source of secreted proteins in adipose tissue . as the adipose tissue came from lean animals , the expected number of macrophages should be low and in fact only three secreted proteins common to the secretomes of whole adipose tissue and human macrophages were found . this leads to the important conclusion that the secretome of whole adipose tissue is not simply the sum of each of its component parts but is the result of a paracrine interaction between the multiple cell types within adipose tissue .
objectivevisceral adipose tissue ( vat ) is more closely linked to insulin resistance than subcutaneous adipose tissue ( sat ) . 
 we conducted a quantitative analysis of the secretomes of vat and sat to identify differences in adipokine secretion that account for the adverse metabolic consequences of vat.research design and methodswe used lectin affinity chromatography followed by comparison of isotope - labeled amino acid incorporation rates to quantitate relative differences in the secretomes of vat and sat explants . because adipose tissue is composed of multiple cell types , which may contribute to depot - specific differences in secretion , we isolated preadipocytes and microvascular endothelial cells ( mvecs ) and compared their secretomes to those from whole adipose tissue.resultsalthough there were no discrete depot - specific differences in the secretomes from whole adipose tissue , preadipocytes , or mvecs , vat exhibited an overall higher level of protein secretion than sat . 
 more proteins were secreted in twofold greater abundance from vat explants compared with sat explants ( 59% versus 21% ) , preadipocytes ( 68% versus 0% ) , and mvecs ( 62% versus 15% ) . 
 the number of proteins in the whole adipose tissue secretome was greater than the sum of its cellular constituents . 
 finally , almost 50% of the adipose tissue secretome was composed of factors with a role in angiogenesis.conclusionsvat has a higher secretory capacity than sat , and this difference is an intrinsic feature of its cellular components . in view of the number of angiogenic factors in the adipose tissue secretome , we propose that vat represents a more readily expandable tissue depot .