twenty - two volunteers ( 10 women ) aged between 18 and 50 years underwent a rigorous protocol using standard measures of obesity and insulin resistance .
specifically , participants were nondiabetic ( normal glucose tolerance or impaired glucose tolerance ) according to an oral glucose tolerance test ( ogtt ) ( world health organization 1999 criteria ) , nonsmokers at the time of the study , and healthy according to a detailed physical examination and routine blood analyses .
no participants had clinical or laboratory signs of acute or chronic infection or took any medication or used illicit drugs at the time of the study .
the protocol was approved by the alfred hospital ethics committee and complied with the declaration of helsinki 2004 .
all participants were characterized for plasma inflammation markers , body composition , glucose tolerance , insulin action , and nf-b and jnk1 activity in pbmcs and muscle and adipose tissue ( table 1 ) .
anthropometric and metabolic parameters of the study population * p < 0.05 male vs. female subjects .
all subjects underwent medical screening , which included history , physical examination , and basic laboratory tests ( including fasting plasma lipid levels , liver function tests , urea , creatinine , and electrolyte levels , urinary illicit drug screening , an anthropometric assessment , an ogtt , and a hyperinsulinemic - euglycemic glucose clamp . before metabolic testing , participants were asked to abstain from strenuous exercise and caffeine for 3 days .
body composition was estimated by total - body dual - energy x - ray absorptiometry ( dpx - l ; lunar radiation , madison , wi ) with calculations of percent body fat , fat mass , and fat - free mass as described previously ( 11 ) .
waist and hip circumferences were measured and waist - to - hip ratio ( whr ) was calculated as an index of body fat distribution .
a 2-h 75-g ogtt was performed after a 12-h overnight fast , and glucose tolerance status was determined by world health organization 1999 criteria .
plasma glucose concentrations were determined by the glucose oxidase method ( elm 105 ; radiometer , copenhagen , denmark ) .
insulin action was assessed at physiological insulin concentrations during a hyperinsulinemic - euglycemic clamp . in brief , after an overnight fast , a primed continuous intravenous insulin infusion ( 9 mu / kg ) was administered for 120 min at a constant rate of 40 mu / mbody surface area per minute ( m ) .
plasma glucose was measured every 5 min during the clamp , and the variable infusion rate of glucose was adjusted to maintain blood glucose at a constant value of 5 mmol / l .
the rate of total insulin - stimulated glucose disposal ( m ) was calculated for the last 40 min of the insulin infusions .
blood samples for analysis of inflammation markers were drawn before the start of the glucose clamp . biopsies of subcutaneous adipose and vastus lateralis muscle were performed on a separate day using standard aseptic technique and local anesthesia . in brief , before a percutaneous muscle biopsy of the vastus lateralis muscle , a scalpel blade was used to make a 7-mm skin incision and to cut the fascia .
a side - cutting muscle biopsy needle was passed through the incision to obtain 100 mg of muscle tissue .
adipose tissue was obtained by needle biopsy from the abdominal area , 12 cm superior to mcburney 's point . after making an 5-mm skin incision ,
the needle biopsy was performed using a 50-ml plastic syringe attached to a 13-gauge aspiration needle .
the muscle and adipose tissues were immediately placed in liquid nitrogen and then stored at 80c .
blood samples were drawn using standard phlebotomy techniques into sodium citrate vacutainers for measurement of inflammatory markers .
the tubes were centrifuged immediately ( 1,500 g , 15 min , 4c ) , and the plasma was stored at 80c until analyses were performed .
monocyte chemoattractant protein-1 ( mcp-1 ) was measured using a beadlyte plex kit ( upstate cell signaling solutions , charlottesville , va ) .
multiplexed assays were run according to the manufacturer 's instructions on a luminex 100 bioplex machine using luminex pro software version 1.7 ( bio - rad , atherton , ca ) . plasma high - sensitivity c - reactive protein ( hscrp ) was measured by an immunoturbidimetric assay .
hepatic enzymes , alanine aminotransferase ( alt ) and -glutamyltranspeptidase ( ggt ) , were measured by nadh ( without p-5-p ) andl--glutamyl-3-carboxy-4-nitroanilide substrates , respectively .
hscrp and hepatic enzymes ( alt and ggt ) were measured by alfred hospital pathology services using the architect system ( abbott diagnostics , abbott park , il ) .
pbmcs were isolated from whole blood using ficoll - paque plus density centrifugation ( amersham biosciences , uppsala , sweden ) .
the pbmc pellet was resuspended in fbs with 10% dmso and stored at 80c . nuclear extraction from pbmcs and adipose and skeletal muscle tissue
muscle and adipose tissue biopsy samples were homogenized in a 1:5 w / v ice - cold homogenization buffer ( 50 mmol / l tris - hcl , ph 7.8 , 10 mmol / l edta , ph 8.0 , 100 mmol / l naf , 2 mmol / l na3 vo4 , 1 mmol / l sodium pyrophosphate , 250 mol / l phenylmethylsulfonyl fluoride , 10 g / ml aprotinin , and 10 g / ml leupeptin ) with a hand - held homogenizer ( polytron - aggregate ; kinematica , switzerland ) .
samples were then rotated for 30 min , end - over - end , at 4c , before being centrifuged ( 14,000 g , 30 min , 4c ) to isolate the nuclear pellet .
the nuclear pellet was then resuspended in 500 l buffer a ( 10 mmol / l hepes , ph 7.9 , 10 mmol / l kci , 0.1 mmol / l edta , 0.1 mmol / l egta , and 0.7% v / v igepal ) , vortexed and centrifuged ( 7,000 g , 1 min , room temperature ) , and then resuspended in 500 l buffer b ( 10 mmol / l hepes , ph 7.9 , 10 mmol / l kci , 0.1 mmol / l edta , and 0.1 mmol / l egta ) .
supernatant was removed , and the pellet was resuspended in 150 l buffer c ( 20 mmol / l hepes , ph 7.9 , 400 mmol / l nacl , 1 mmol / l edta , and 1 mmol / l egta ) , incubated on ice for 20 min , and then centrifuged ( 10,000 g , 30 min , room temperature ) .
pbmc samples were washed in excess pbs and centrifuged ( 400 g , 5 min , 4c ) , to remove residual fbs and dmso .
the supernatant was discarded , and the pellets were resuspended in 100 l triple detergent buffer ( 50 mmol / l tris - hcl , ph 8.0 , 150 mmol / l nacl , 0.02% nan3 , 0.1% sds , 100 g / ml phenylmethylsulfonyl fluoride , 1 g / ml aprotinin , 1% nonidet p-40 , and 0.5% sodium deoxycholate , in milli - q h2 o ) .
these were then sonicated for 1 min and centrifuged ( 400 g , 3 min , 4c ) .
the protein concentration of all protein isolates was determined using the bicinchoninic acid protein assay ( pierce , rockford , il ) , performed according to the manufacturer 's instructions .
the transam nfb dna - binding activity assay ( active motif , carlsbad , ca ) was used to detect and quantify nf-b transcription factor activation , specifically of the p65 subunit .
nuclear extracts obtained from muscle ( 1 g protein / well ) and adipose tissue ( 5 g protein / well ) biopsies and pbmc ( 30 g protein / well ) samples were analyzed for their binding capacity to an nf-b consensus sequence in labeled dna in a 96-well plate format .
the assays were performed according to the manufacturer 's instructions , and absorbance was measured on a victor3v multilabel plate reader ( perkinelmer , wellesley , ma ) .
phosphorylation of jnk ( [ thr / tyr ] /total jnk ) was used as a surrogate for jnk1/2 activity as described previously ( 21 ) .
pbmc , subcutaneous adipose tissue , and skeletal muscle samples were homogenized , and protein concentrations were determined .
total protein ( 40 g ) was separated by sds - page and blotted onto a polyvinylidene fluoride membrane .
blots were performed for phosphorylated jnk ( 1:1,000 ) and total jnk ( 1:1,000 ) using antibodies from cell signaling technologies .
bands were visualized using a bio - rad anti - rabbit igg horseradish peroxidase ( 1:4,000 ) conjugated antibody and ecl solution ( sigma ) and developed on film .
jnk1/2 activity in muscle and adipose tissue was available for only 18 individuals because insufficient tissue was available for analyses .
gene expression of the p65 subunit of nf-b and ib in pbmcs was assessed by real - time quantitative rt - pcr , performed using the taqman system based on real - time detection of accumulated fluorescence ( abi prism 7700 sequence detection system ; pe biosystems , foster city , ca ) as described previously ( 23 ) .
briefly , 4 g total rna was isolated from pbmcs with trizol according to the manufacturer 's instructions ( gibco brl , grand island , ny ) .
the resulting rna was used to synthesize cdna with a superscript first strand synthesis system ( gibco brl ) . to control for variation in the amount of dna available for pcr in the different samples , gene expression was normalized simultaneously ( by multiplexing ) in relation to the expression of the housekeeping gene 18s rrna ( 18s rrna taqman control reagent kit ) as an endogenous control .
primers and taqman probes for the proteins described above were constructed with the help of primer express ( perkinelmer ) ( for sequences , see table 2 ) and fluorescence for each cycle was quantitatively analyzed by an abi prism 7700 sequence detection system .
results are expressed as arbitrary units , related to minor groove binding ( mgb ) probe sample fluorescence , corrected to endogenous 18s expression .
real - time rt - pcr probes and primers used for amplification of nf-b p65 and ib in pbmcs statistical analyses were performed using sas jump statistics software ( sas institute , cary , nc ) .
the values for inflammatory markers and m were logarithmically transformed before analysis to approximate normal distributions .
the relationships between inflammatory markers , nf-b , jnk1/2 , and anthropometric and metabolic variables were examined by calculating pearson correlation coefficients , and p values were corrected for multiple comparisons ( benjamini - hochberg procedure ) .
multiple linear regression models and partial correlations were used to examine the relationships after adjustment for covariates .
stepwise regression was used to determine a relative contribution of each of the variables to the model .
our correlation tests are sufficiently sensitive at  = 0.05 with a power of 1   = 0.80 for two - sided correlations at r = 0.53 and for one - sided correlations at r = 0.48 ( n = 22 , degrees of freedom = 20 ) .
because inflammation markers are known to be positively associated with obesity and negatively with insulin sensitivity , the one - sided calculation is most relevant to the current analyses .
post hoc statistical power calculations for the multiple regression models of our main findings , which all had r>0.6 , give power estimates greater than 0.90 for up to five independent variables in the model at  = 0.05 .
body composition was estimated by total - body dual - energy x - ray absorptiometry ( dpx - l ; lunar radiation , madison , wi ) with calculations of percent body fat , fat mass , and fat - free mass as described previously ( 11 ) .
waist and hip circumferences were measured and waist - to - hip ratio ( whr ) was calculated as an index of body fat distribution .
a 2-h 75-g ogtt was performed after a 12-h overnight fast , and glucose tolerance status was determined by world health organization 1999 criteria .
plasma glucose concentrations were determined by the glucose oxidase method ( elm 105 ; radiometer , copenhagen , denmark ) .
insulin action was assessed at physiological insulin concentrations during a hyperinsulinemic - euglycemic clamp . in brief , after an overnight fast , a primed continuous intravenous insulin infusion ( 9 mu / kg ) was administered for 120 min at a constant rate of 40 mu / mbody surface area per minute ( m ) .
plasma glucose was measured every 5 min during the clamp , and the variable infusion rate of glucose was adjusted to maintain blood glucose at a constant value of 5 mmol / l .
the rate of total insulin - stimulated glucose disposal ( m ) was calculated for the last 40 min of the insulin infusions .
blood samples for analysis of inflammation markers were drawn before the start of the glucose clamp .
biopsies of subcutaneous adipose and vastus lateralis muscle were performed on a separate day using standard aseptic technique and local anesthesia . in brief , before a percutaneous muscle biopsy of the vastus lateralis muscle , a scalpel blade was used to make a 7-mm skin incision and to cut the fascia .
a side - cutting muscle biopsy needle was passed through the incision to obtain 100 mg of muscle tissue .
adipose tissue was obtained by needle biopsy from the abdominal area , 12 cm superior to mcburney 's point . after making an 5-mm skin incision
, the needle biopsy was performed using a 50-ml plastic syringe attached to a 13-gauge aspiration needle .
the muscle and adipose tissues were immediately placed in liquid nitrogen and then stored at 80c .
blood samples were drawn using standard phlebotomy techniques into sodium citrate vacutainers for measurement of inflammatory markers .
the tubes were centrifuged immediately ( 1,500 g , 15 min , 4c ) , and the plasma was stored at 80c until analyses were performed .
monocyte chemoattractant protein-1 ( mcp-1 ) was measured using a beadlyte plex kit ( upstate cell signaling solutions , charlottesville , va ) .
multiplexed assays were run according to the manufacturer 's instructions on a luminex 100 bioplex machine using luminex pro software version 1.7 ( bio - rad , atherton , ca ) . plasma high - sensitivity c - reactive protein ( hscrp ) was measured by an immunoturbidimetric assay .
hepatic enzymes , alanine aminotransferase ( alt ) and -glutamyltranspeptidase ( ggt ) , were measured by nadh ( without p-5-p ) andl--glutamyl-3-carboxy-4-nitroanilide substrates , respectively .
hscrp and hepatic enzymes ( alt and ggt ) were measured by alfred hospital pathology services using the architect system ( abbott diagnostics , abbott park , il ) .
pbmcs were isolated from whole blood using ficoll - paque plus density centrifugation ( amersham biosciences , uppsala , sweden ) .
the pbmc pellet was resuspended in fbs with 10% dmso and stored at 80c . nuclear extraction from pbmcs and adipose and skeletal muscle tissue
briefly , muscle and adipose tissue biopsy samples were homogenized in a 1:5 w / v ice - cold homogenization buffer ( 50 mmol / l tris - hcl , ph 7.8 , 10 mmol / l edta , ph 8.0 , 100 mmol / l naf , 2 mmol / l na3 vo4 , 1 mmol / l sodium pyrophosphate , 250 mol / l phenylmethylsulfonyl fluoride , 10 g / ml aprotinin , and 10 g / ml leupeptin ) with a hand - held homogenizer ( polytron - aggregate ; kinematica , switzerland ) .
samples were then rotated for 30 min , end - over - end , at 4c , before being centrifuged ( 14,000 g , 30 min , 4c ) to isolate the nuclear pellet .
the nuclear pellet was then resuspended in 500 l buffer a ( 10 mmol / l hepes , ph 7.9 , 10 mmol / l kci , 0.1 mmol / l edta , 0.1 mmol / l egta , and 0.7% v / v igepal ) , vortexed and centrifuged ( 7,000 g , 1 min , room temperature ) , and then resuspended in 500 l buffer b ( 10 mmol / l hepes , ph 7.9 , 10 mmol / l kci , 0.1 mmol / l edta , and 0.1 mmol / l egta ) .
supernatant was removed , and the pellet was resuspended in 150 l buffer c ( 20 mmol / l hepes , ph 7.9 , 400 mmol / l nacl , 1 mmol / l edta , and 1 mmol / l egta ) , incubated on ice for 20 min , and then centrifuged ( 10,000 g , 30 min , room temperature ) .
pbmc samples were washed in excess pbs and centrifuged ( 400 g , 5 min , 4c ) , to remove residual fbs and dmso .
the supernatant was discarded , and the pellets were resuspended in 100 l triple detergent buffer ( 50 mmol / l tris - hcl , ph 8.0 , 150 mmol / l nacl , 0.02% nan3 , 0.1% sds , 100 g / ml phenylmethylsulfonyl fluoride , 1 g / ml aprotinin , 1% nonidet p-40 , and 0.5% sodium deoxycholate , in milli - q h2 o ) .
these were then sonicated for 1 min and centrifuged ( 400 g , 3 min , 4c ) .
the protein concentration of all protein isolates was determined using the bicinchoninic acid protein assay ( pierce , rockford , il ) , performed according to the manufacturer 's instructions .
the transam nfb dna - binding activity assay ( active motif , carlsbad , ca ) was used to detect and quantify nf-b transcription factor activation , specifically of the p65 subunit .
nuclear extracts obtained from muscle ( 1 g protein / well ) and adipose tissue ( 5 g protein / well ) biopsies and pbmc ( 30 g protein / well ) samples were analyzed for their binding capacity to an nf-b consensus sequence in labeled dna in a 96-well plate format .
the assays were performed according to the manufacturer 's instructions , and absorbance was measured on a victor3v multilabel plate reader ( perkinelmer , wellesley , ma ) .
phosphorylation of jnk ( [ thr / tyr ] /total jnk ) was used as a surrogate for jnk1/2 activity as described previously ( 21 ) .
pbmc , subcutaneous adipose tissue , and skeletal muscle samples were homogenized , and protein concentrations were determined .
total protein ( 40 g ) was separated by sds - page and blotted onto a polyvinylidene fluoride membrane .
blots were performed for phosphorylated jnk ( 1:1,000 ) and total jnk ( 1:1,000 ) using antibodies from cell signaling technologies .
bands were visualized using a bio - rad anti - rabbit igg horseradish peroxidase ( 1:4,000 ) conjugated antibody and ecl solution ( sigma ) and developed on film .
jnk1/2 activity in muscle and adipose tissue was available for only 18 individuals because insufficient tissue was available for analyses .
gene expression of the p65 subunit of nf-b and ib in pbmcs was assessed by real - time quantitative rt - pcr , performed using the taqman system based on real - time detection of accumulated fluorescence ( abi prism 7700 sequence detection system ; pe biosystems , foster city , ca ) as described previously ( 23 ) .
briefly , 4 g total rna was isolated from pbmcs with trizol according to the manufacturer 's instructions ( gibco brl , grand island , ny ) .
the resulting rna was used to synthesize cdna with a superscript first strand synthesis system ( gibco brl ) . to control for variation in the amount of dna available for pcr in the different samples , gene expression was normalized simultaneously ( by multiplexing ) in relation to the expression of the housekeeping gene 18s rrna ( 18s rrna taqman control reagent kit ) as an endogenous control .
primers and taqman probes for the proteins described above were constructed with the help of primer express ( perkinelmer ) ( for sequences , see table 2 ) and fluorescence for each cycle was quantitatively analyzed by an abi prism 7700 sequence detection system .
results are expressed as arbitrary units , related to minor groove binding ( mgb ) probe sample fluorescence , corrected to endogenous 18s expression .
real - time rt - pcr probes and primers used for amplification of nf-b p65 and ib in pbmcs
statistical analyses were performed using sas jump statistics software ( sas institute , cary , nc ) .
the values for inflammatory markers and m were logarithmically transformed before analysis to approximate normal distributions .
the relationships between inflammatory markers , nf-b , jnk1/2 , and anthropometric and metabolic variables were examined by calculating pearson correlation coefficients , and p values were corrected for multiple comparisons ( benjamini - hochberg procedure ) .
multiple linear regression models and partial correlations were used to examine the relationships after adjustment for covariates .
stepwise regression was used to determine a relative contribution of each of the variables to the model .
differences between men and women were assessed by unpaired student 's t test . statistical significance was assumed when p < 0.05 .
our correlation tests are sufficiently sensitive at  = 0.05 with a power of 1   = 0.80 for two - sided correlations at r = 0.53 and for one - sided correlations at r = 0.48 (
because inflammation markers are known to be positively associated with obesity and negatively with insulin sensitivity , the one - sided calculation is most relevant to the current analyses .
post hoc statistical power calculations for the multiple regression models of our main findings , which all had r>0.6 , give power estimates greater than 0.90 for up to five independent variables in the model at  = 0.05 .
all subjects were deemed healthy according to their physical examination , were normotensive with normal lipid profile , full blood count , plasma crp levels , liver function tests ( table 1 ) , and renal function ( estimated glomerular filtration rate > 60 ml / min per m ) , and therefore subclinical inflammation was ruled out .
women had higher percent body fat and hdl cholesterol levels , but there were no other sex differences seen among any of the other anthropometric and metabolic variables .
nf-b activity in pbmcs was positively associated with nf-b activity in the skeletal muscle ( r = 0.68 , p = 0.0004 ) but not in adipose tissue ( r = 0.17 , p = 0.4 ) .
jnk1/2 activity in pbmcs was not related to jnk1/2 activity in skeletal muscle ( r = 0.14 , p = 0.6 ) or subcutaneous adipose tissue ( r = 0.34 , p = 0.2 ) .
in addition , no relationship between jnk1/2 activity and nf-b activity was identified in any of the tissues studied ( all p > 0.2 ) .
white blood cell count ( r = 0.39 , p < 0.05 ) and plasma levels of mcp-1 ( r = 0.63 , p < 0.05 ) were each associated with nf-b activity in pbmcs but not with nf-b activity in the skeletal muscle or subcutaneous adipose tissue ( table 3 ) .
plasma hscrp ( table 3 ) was not related to nf-b activity in any of the tissues investigated and did not correlate with insulin sensitivity ( r = 0.20 , p = 0.4 ) .
jnk1/2 activity was not associated with any of these markers of inflammation ( p > 0.1 ) .
pearson correlation coefficients between nf-b and jnk1/2 activity in pbmcs and muscle and adipose tissue with anthropometric and metabolic variables * p < 0.05 .
nf-b activity in either pbmcs or skeletal muscle was not associated with an overall measure of obesity , percent body fat ( table 3,fig . 1 ) .
nf-b activity in subcutaneous adipose tissue also was not related to any of the anthropometric variables .
furthermore , the gene expression of ib and nf-b ( p65 ) in pbmc did not correlate with any of the anthropometric or metabolic parameters or nf-b activity in pbmcs ( data not shown ) .
however , we found that nf-b activity in both pbmcs and skeletal muscle was positively associated with whr ( r = 0.48 , p = 0.02 ; r = 0.68 , p = 0.003 ) ( table 3,fig .
1 ) before and after adjustment for age and sex ( both p < 0.05 )
. this association persisted after additional adjustment for percent body fat ( both p < 0.05 ) .
tissue jnk1/2 activity was not related to any measures of obesity ( both p > 0.1 ) ( table 3 ) .
nf-b p65 activity in pbmcs and central obesity , insulin sensitivity , and jnk1 activity in subcutaneous adipose tissue and insulin sensitivity .
c : nf-b p65 activity in pbmcs and m. d : jnk1/2 activity and insulin sensitivity ( m ) .
nf-b activity in pbmcs was inversely associated with insulin sensitivity before ( r = 0.46 , p = 0.03 ) ( table 3 ) and after adjustment for age , sex , percent body fat , and whr ( p = 0.02 ) . in stepwise regression ,
elevated nf-b activity in pbmcs explained 16% of the variance in the model after adjustment for the variables stated above .
nf-b activity in skeletal muscle and adipose tissue were not associated with insulin sensitivity ( both p > 0.1 ) .
jnk1/2 activity in the adipose tissue was related to insulin sensitivity ( r = 0.54 , p < 0.05 ) ( fig .
2 ) before and after adjustment for age , sex , percent body fat , and whr ( p = 0.04 ) . in stepwise regression with age , sex , percent body fat , and whr , jnk1/2 activity in adipose tissue explained the 29% variance in insulin sensitivity ( p = 0.02 ) . when both jnk1/2 in adipose tissue and nf-b activity in pbmcs were entered in the same model , only jnk1/2 activity in adipose tissue remained a significant determinant of insulin sensitivity , explaining 29% ( p = 0.02 ) , whereas percent body fat contributed 21% ( p = 0.02 ) and sex 10% ( p = 0.07 ) . when forced into this model , nf-b activity in pbmcs explained only 4% of the variance in insulin sensitivity ( p = 0.1 ) .
jnk1/2 activity in pbmcs and muscle was not associated with any measures of obesity or glucose metabolism ( all p > 0.2 ) .
exclusion of the two subjects with impaired glucose metabolism ( one with impaired glucose tolerance and one with impaired fasting glucose ) did not significantly alter the correlations presented .
representative immunoblots of human adipose tissue ( n = 6 ) probed for total jnk ( tjnk ) and phosphorylated jnk ( pjnk , thr / tyr ) .
nf-b activity in pbmcs was positively associated with nf-b activity in the skeletal muscle ( r = 0.68 , p = 0.0004 ) but not in adipose tissue ( r = 0.17 , p = 0.4 ) .
jnk1/2 activity in pbmcs was not related to jnk1/2 activity in skeletal muscle ( r = 0.14 , p = 0.6 ) or subcutaneous adipose tissue ( r = 0.34 , p = 0.2 ) .
in addition , no relationship between jnk1/2 activity and nf-b activity was identified in any of the tissues studied ( all p > 0.2 ) .
white blood cell count ( r = 0.39 , p < 0.05 ) and plasma levels of mcp-1 ( r = 0.63 , p < 0.05 ) were each associated with nf-b activity in pbmcs but not with nf-b activity in the skeletal muscle or subcutaneous adipose tissue ( table 3 ) .
plasma hscrp ( table 3 ) was not related to nf-b activity in any of the tissues investigated and did not correlate with insulin sensitivity ( r = 0.20 , p = 0.4 ) .
jnk1/2 activity was not associated with any of these markers of inflammation ( p > 0.1 ) .
pearson correlation coefficients between nf-b and jnk1/2 activity in pbmcs and muscle and adipose tissue with anthropometric and metabolic variables * p < 0.05 .
nf-b activity in either pbmcs or skeletal muscle was not associated with an overall measure of obesity , percent body fat ( table 3,fig
nf-b activity in subcutaneous adipose tissue also was not related to any of the anthropometric variables .
furthermore , the gene expression of ib and nf-b ( p65 ) in pbmc did not correlate with any of the anthropometric or metabolic parameters or nf-b activity in pbmcs ( data not shown ) .
however , we found that nf-b activity in both pbmcs and skeletal muscle was positively associated with whr ( r = 0.48 , p = 0.02 ; r = 0.68 , p = 0.003 ) ( table 3,fig .
1 ) before and after adjustment for age and sex ( both p < 0.05 )
. this association persisted after additional adjustment for percent body fat ( both p < 0.05 ) .
tissue jnk1/2 activity was not related to any measures of obesity ( both p > 0.1 ) ( table 3 ) .
nf-b p65 activity in pbmcs and central obesity , insulin sensitivity , and jnk1 activity in subcutaneous adipose tissue and insulin sensitivity .
c : nf-b p65 activity in pbmcs and m. d : jnk1/2 activity and insulin sensitivity ( m ) .
nf-b activity in pbmcs was inversely associated with insulin sensitivity before ( r = 0.46 , p = 0.03 ) ( table 3 ) and after adjustment for age , sex , percent body fat , and whr ( p = 0.02 ) . in stepwise regression ,
elevated nf-b activity in pbmcs explained 16% of the variance in the model after adjustment for the variables stated above .
nf-b activity in skeletal muscle and adipose tissue were not associated with insulin sensitivity ( both p > 0.1 ) .
jnk1/2 activity in the adipose tissue was related to insulin sensitivity ( r = 0.54 , p < 0.05 ) ( fig .
2 ) before and after adjustment for age , sex , percent body fat , and whr ( p = 0.04 ) . in stepwise regression with age , sex , percent body fat , and whr , jnk1/2 activity in adipose tissue explained the 29% variance in insulin sensitivity ( p = 0.02 ) . when both jnk1/2 in adipose tissue and nf-b activity in pbmcs were entered in the same model , only jnk1/2 activity in adipose tissue remained a significant determinant of insulin sensitivity , explaining 29% ( p = 0.02 ) , whereas percent body fat contributed 21% ( p = 0.02 ) and sex 10% ( p = 0.07 ) . when forced into this model , nf-b activity in pbmcs explained only 4% of the variance in insulin sensitivity ( p = 0.1 ) .
jnk1/2 activity in pbmcs and muscle was not associated with any measures of obesity or glucose metabolism ( all p > 0.2 ) .
exclusion of the two subjects with impaired glucose metabolism ( one with impaired glucose tolerance and one with impaired fasting glucose ) did not significantly alter the correlations presented .
representative immunoblots of human adipose tissue ( n = 6 ) probed for total jnk ( tjnk ) and phosphorylated jnk ( pjnk , thr / tyr ) .
in the present study , we showed for the first time that , among healthy , nondiabetic individuals , both nf-b activity in pbmcs and jnk1/2 activity in subcutaneous adipose tissue were important determinants of insulin resistance and explained 16 and 29% , respectively , of its variance after adjustment for appropriate covariates .
moreover , nf-b activity in both pbmcs and skeletal muscle was also associated with central obesity .
the cross - sectional relationships between nf-b activity in pbmcs and adiposity were consistent with previous findings in obese patients in whom nf-b activity in pbmcs was elevated ( 20,24 ) and correlated with bmi ( 20,25 ) .
we added to the current evidence by showing this relationship across a wide range of adiposity .
we also provided more precise measures of overall adiposity , specifically percent body fat by dual - energy x - ray absorptiometry as opposed to bmi , but , interestingly , our study showed that nf-b activity in pbmcs was specifically associated with central obesity but not overall adiposity ( measured as total percent body fat or bmi ) , perhaps because we have studied participants with a wide range of adiposity as opposed to only morbidly obese patients . in the above - mentioned studies ( 20,24 ) ,
it is well established that central adiposity is a stronger predictor than overall adiposity of insulin resistance and progression to type 2 diabetes than overall adiposity and is likely to be responsible for the proinflammatory phenotype seen in these conditions ( 2631 ) .
thus , our additional findings showed for the first time a relationship between nf-b activity in skeletal muscle and central obesity , suggesting that activation of the nf-b / ib pathway in muscle may contribute to a local inflammatory environment due to lipid accumulation and contributing to insulin resistance .
in accordance with two other studies ( 20,24 ) , we also found a relationship between increased nf-b activity in pbmcs and insulin resistance .
in contrast to these studies , which used a calculated homa as a proxy for insulin resistance , we examined insulin resistance directly using the standard hyperinsulinemic - euglycemic clamp . moreover , we showed that the relationship between nf-b activity in pbmcs and insulin resistance is independent of the degree of obesity and explained 16% of variance in insulin resistance after adjustment for age , sex , percent body fat , and whr . although previous studies have shown that nf-b activation in skeletal muscle is related to insulin resistance in obese humans ( 20,24 ) , this was not seen in our study in nondiabetic healthy individuals .
our data are consistent with rodent studies in which overexpression of the p65 subunit of nf-b in skeletal muscle had no effect on insulin resistance as measured by glucose clamp ( 32 ) . in our study
, we also found no relationship between activation of nf-b in adipose tissue and any of the anthropometric or metabolic parameters .
animal models of type 2 diabetes have demonstrated an association between insulin resistance and elevated levels of the activator of nf-b , ikk , at the gene level , in perigonadal but not in mesenteric adipose tissue ( 33 ) .
gene levels of the inhibitor of nf-b ( ib ) have also been associated with central adiposity in obese elderly women ( 34 ) . both of these studies ( 33,34 ) , however , examined only gene expression as opposed to activity .
the absence of an association between nf-b activity in adipose tissue and anthropometric or metabolic parameters in the present study could also be due to differences in the populations examined .
both of the previously reported studies described the relationship in obese and insulin - resistant animals / populations ( 33,34 ) , whereas our study was conducted in a healthy population .
interestingly , nf-b activity in pbmcs was associated with circulating mcp-1 levels but not with crp .
mcp-1 is one of the many downstream cytokine targets of the key proinflammatory transcription factor , nf-b .
mcp-1 is known to recruit white blood cells to the site of inflammation and therefore may be considered an important driver and perpetuator of nf-b activation and inflammation ( 35 ) .
nf-b activity in pbmcs and adipose tissue have been shown previously to correlate with the gene ( 20,34 ) and protein ( 20 ) expression of interleukin-6 , macrophage inhibitory factor , and hscrp in obese individuals . in our study , however , plasma levels of crp were not associated with either nf-b activity or insulin sensitivity probably because of the low plasma levels of crp ( all values within normal range ) in our healthy nondiabetic population .
therefore , nf-b may be a more sensitive early marker of inflammation than crp in healthy individuals .
we have made the novel observation that nf-b activity in pbmcs is an important determinant of both central adiposity and insulin resistance .
interestingly , arkan et al . ( 13 ) demonstrated that mice lacking ikk in pbmcs , which makes them unable to activate the nf-b / ib pathway , are protected from systemic insulin resistance .
our data demonstrated this relationship for the first time in humans . to date , jnk1/2 activity has only been associated with obesity in animal models of obesity and diabetes ( 9 ) . in this study , we showed that jnk1/2 activity in pbmcs , skeletal muscle , and subcutaneous adipose tissue was not associated with any measure of obesity in humans .
importantly , in the current study we also showed that jnk1/2 activity in subcutaneous adipose tissue was associated with insulin resistance , independently of age , sex , percent body fat , and whr and explained 29% of the variance in insulin resistance after adjustment for these covariates . in this study
, we have made the novel observation that when both nf-b activity in pbmcs and jnk1/2 activity in subcutaneous adipose tissue are included in this model , only jnk1/2 activity was a significant determinant of insulin resistance , suggesting not only that these pathways are codependent but also that jnk1/2 activity is an independent and therefore potentially more important determinant of insulin resistance in nondiabetic , otherwise healthy individuals .
jnk activity in skeletal muscle has been shown to be associated with insulin resistance in obese and diabetic subjects ( 22 ) .
we have also shown previously that obese insulin - resistant humans ( insulin sensitivity assessed by homa ) have increased jnk1/2 phosphorylation in skeletal muscle ( 21 ) . in our present study , however , we did not see a relationship between jnk1/2 activity in skeletal muscle and insulin resistance , most likely because of our different study population , i.e. , healthy nondiabetic individuals versus obese , type 2 diabetic subjects .
adipose tissue jnk activity was not examined in either of these previous studies ( 21,22 ) , so we can not compare our results in this regard .
it could be hypothesized that different inflammatory pathways may be important in the initiation of insulin resistance as opposed to when diabetes has already developed . in our study ,
tissue jnk1/2 activity was not associated with circulating inflammatory makers , suggesting that tissue - specific inflammation and activation of jnk are enough to cause insulin resistance .
first , because of the cross - sectional nature of the study , we can not delineate the cause and effect relationships between jnk and nf-b activity and insulin resistance .
second , the sample size is small , and , hence , the results should be interpreted with caution .
however , the strength of the correlations and their robustness in various adjusted models attests to the validity of our findings .
third , standard imaging methods such as computed tomography or magnetic resonance imaging would have provided a better assessment of central adiposity than waist circumference . in conclusion , we have demonstrated that elevated nf-b activity in pbmcs and jnk1/2 activity in subcutaneous adipose tissue are both important determinants of insulin resistance in a healthy nondiabetic population .
moreover , we show that jnk1/2 activity in adipose tissue but not nf-b activity in pbmc is an independent determinant of insulin resistance in this population .
further investigation is warranted to determine the mechanisms by which changes in nf-b and jnk1/2 activity contribute to the development of type 2 diabetes in humans .
 objectivechronic low - grade activation of the immune system ( clais ) predicts type 2 diabetes via a decrease in insulin sensitivity . 
 our study investigated potential relationships between nuclear factor-b ( nf-b ) and c - jun nh2-terminal kinase ( jnk ) pathways  two pathways proposed as the link between clais and insulin resistance.research design and methodsadiposity ( dual - energy x - ray absorptiometry ) , waist - to - hip ratio ( whr ) , and insulin sensitivity ( m , hyperinsulinemic - euglycemic clamp ) were measured in 22 healthy nondiabetic volunteers ( aged 29  11 years , body fat 28  11% ) . 
 nf-b activity ( dna - binding assay ) and jnk1/2 activity ( phosphorylated jnk ) were assessed in biopsies of the vastus lateralis muscle and subcutaneous adipose tissue and in peripheral blood mononuclear cell ( pbmc ) lysates.resultsnf-b activities in pbmcs and muscle were positively associated with whr after adjustment for age , sex , and percent body fat ( both p < 0.05 ) . 
 nf-b activity in pbmcs was inversely associated with m after adjustment for age , sex , percent body fat , and whr ( p = 0.02 ) and explained 16% of the variance of m. there were no significant relationships between nf-b activity and m in muscle or adipose tissue ( both ns ) . 
 adipose - derived jnk1/2 activity was not associated with obesity ( all p > 0.1 ) , although it was inversely related to m ( r = 0.54 , p < 0.05 ) and explained 29% of its variance . 
 when both nf-b and jnk1/2 were examined statistically , only jnk1/2 activity in adipose tissue was a significant determinant of insulin resistance ( p = 0.02).conclusionsjnk1/2 activity in adipose tissue but not nf-b activity in pbmcs is an independent determinant of insulin resistance in healthy individuals . 