study subjects were participants of the korean sarcopenic obesity study , an ongoing prospective , observational , cohort study .
the details of the study design and objectives have been published previously ( 10,11 ) .
none of the participants had a history of cardiovascular disease ( myocardial infarction , unstable angina , stroke , or cardiovascular revascularization ) , stage 2 hypertension ( resting blood pressure 160/100 mmhg ) , malignancy , or severe renal or hepatic disease .
a prospective study was performed to examine the effect of a 3-month combined exercise program on circulating ctrp-3 and ctrp-5 concentrations in obese women .
women between 30 and 60 years of age with a bmi > 25 kg / m were recruited for the exercise study .
all were otherwise healthy and had a stable body weight ( < 2 kg change in weight in the past 6 months ) and a sedentary lifestyle ( < 20 min of exercise twice a week ) .
women who smoked cigarettes or had diabetes , cardiovascular disease , or chronic kidney or liver disease ; were pregnant or breastfeeding ; had any other major illnesses ; or were taking medication that could have affected laboratory test results were excluded .
the ethics committee of the study institution , in accordance with the world medical association declaration of helsinki , approved the study .
participants were encouraged to train five times a week , with a training session consisting of a brief warm - up , 45 min of aerobic exercise at an intensity of 6075% of the age - predicted maximum heart rate ( 300 kcal / day ) and a 20-min muscle strength training program ( 100 kcal / day ) , and a brief cooldown .
the training program started at 40% of the observed maximal heart rate and gradually increased to 6075% of the maximal heart rate by week 12 .
aerobic exercise included treadmill walking / running ( m901 t ; motus co. , seoul , korea ) and cycling ( cy8866r ; sunny co. , seoul , korea ) .
bmi was calculated as kilograms body weight over height ( in meters ) squared , and waist circumference was measured at the midpoint between the lower border of the rib cage and iliac crest .
all blood samples were obtained in the morning after an 8-h overnight fast and were immediately stored at 70c for subsequent assays .
levels of high - sensitivity c - reactive protein ( hscrp ) and serum insulin were measured with two different electrochemiluminescence immunoassays ( daiichi pure chemicals co. , tokyo , japan ; roche diagnostics , basel , switzerland ) .
homeostasis model assessment of insulin resistance ( homa - ir ) was calculated according to the following equation : fasting insulin ( u / l )  fasting glucose ( nmol / l ) / 22.5 ( 12 ) .
( incheon , korea ) with respect to having the following assay parameters : rbp4 , intra - assay coefficient of variance ( cv ) ( 1.73.7% ) and interassay cv ( 7.08.8% ) ; adiponectin , intra - assay cv ( 2.93.8% ) and interassay cv ( 2.85.5% ) ; and competitive ctrp-3 , intra - assay cv ( 6.18.3% ) and the interassay cv ( 2.48.2% ) .
rbp4 elisa was established with the use of a human rbp4-specific iga monoclonal antibody in a sandwich format , giving rise to comparable recovery of human serum or plasma rbp4 to the one determined by quantitative western blot . to avoid potential cross - reactivity with other ctrp family members ,
a cdna sequence encoding the nh2-terminal half of human ctrp-5 was amplified and cloned into pet21a+ ( novagen , madison , wi ) .
a polyclonal antibody was produced by immunizing rabbits with the nh2-terminal half ctrp-5 protein antigen .
the mature ctrp-5 protein was used for elisa standard as well as for an immobilized antigen .
fifty microliters of the human serum in 1:2 dilutions together with the polyclonal antibody was applied to wells followed by the addition of 50 l of the standard or diluted sample and 50 l of a detection antibody and incubated for 1 h at 37c .
the assay sensitivity was 1 ng / ml . although the degree of precision of the elisa system in terms of intra - assay cv was between 4.0 and 10.0% , interassay cv was between 6.3 and 9.0% .
specificity was determined by human adiponectin , ctrp-1 , ctrp-2 , ctrp-3 , ctrp-6 , ctrp-7 , ctrp-9 , ctrp-10 , ctrp-11 , and ctrp-13 . no cross - reactivity was noted .
the same holds true for > 20 adipokines or metabolic hormones ( data not shown ) .
visceral and subcutaneous fat area was quantified by ct scan ( brilliance 64 ; philips medical systems , cleveland , oh ) .
visceral fat area ( vfa ) , total abdominal fat area , and subcutaneous fat area ( sfa ) were calculated from a 10-cm ct slice image between the fourth and fifth lumbar vertebrae that was obtained during suspended respiration .
ankle - brachial index and brachial - ankle pulse wave velocity ( bapwv ) were measured with a colin waveform analyzer ( model bp-203rpe ii ; nippon colin , komaki , japan ) .
data are mean  sd or median ( interquartile range [ 2575% ] ) . differences between groups were tested with an independent two - sample t test or mann - whitney u test for continuous variables , and differences in the distribution of categorical variables were tested with a  test .
spearman correlation test was performed to determine the relationships of ctrp-3 or ctrp-5 levels with other study variables .
multiple stepwise regression analyses with ctrp-3 or ctrp-5 as the dependent variable were performed to identify the independently associated factors in study participants .
differences between before and after the 3-month combined aerobic and resistance exercise program were tested with paired t test or wilcoxon signed rank test .
data were analyzed with sas version 9.2 ( sas institute , cary , nc ) statistical software .
participants were encouraged to train five times a week , with a training session consisting of a brief warm - up , 45 min of aerobic exercise at an intensity of 6075% of the age - predicted maximum heart rate ( 300 kcal / day ) and a 20-min muscle strength training program ( 100 kcal / day ) , and a brief cooldown .
the training program started at 40% of the observed maximal heart rate and gradually increased to 6075% of the maximal heart rate by week 12 .
aerobic exercise included treadmill walking / running ( m901 t ; motus co. , seoul , korea ) and cycling ( cy8866r ; sunny co. , seoul , korea ) .
bmi was calculated as kilograms body weight over height ( in meters ) squared , and waist circumference was measured at the midpoint between the lower border of the rib cage and iliac crest .
all blood samples were obtained in the morning after an 8-h overnight fast and were immediately stored at 70c for subsequent assays .
levels of high - sensitivity c - reactive protein ( hscrp ) and serum insulin were measured with two different electrochemiluminescence immunoassays ( daiichi pure chemicals co. , tokyo , japan ; roche diagnostics , basel , switzerland ) .
homeostasis model assessment of insulin resistance ( homa - ir ) was calculated according to the following equation : fasting insulin ( u / l )  fasting glucose ( nmol / l ) / 22.5 ( 12 ) .
ag-45a-0035 ) , adiponectin ( cat . # ag-45a-0001 ) , and competitive ctrp-3 ( cat . #
( incheon , korea ) with respect to having the following assay parameters : rbp4 , intra - assay coefficient of variance ( cv ) ( 1.73.7% ) and interassay cv ( 7.08.8% ) ; adiponectin , intra - assay cv ( 2.93.8% ) and interassay cv ( 2.85.5% ) ; and competitive ctrp-3 , intra - assay cv ( 6.18.3% ) and the interassay cv ( 2.48.2% ) .
rbp4 elisa was established with the use of a human rbp4-specific iga monoclonal antibody in a sandwich format , giving rise to comparable recovery of human serum or plasma rbp4 to the one determined by quantitative western blot .
to avoid potential cross - reactivity with other ctrp family members , a cdna sequence encoding the nh2-terminal half of human ctrp-5 was amplified and cloned into pet21a+ ( novagen , madison , wi ) .
a polyclonal antibody was produced by immunizing rabbits with the nh2-terminal half ctrp-5 protein antigen .
the mature ctrp-5 protein was used for elisa standard as well as for an immobilized antigen .
fifty microliters of the human serum in 1:2 dilutions together with the polyclonal antibody was applied to wells followed by the addition of 50 l of the standard or diluted sample and 50 l of a detection antibody and incubated for 1 h at 37c .
the assay sensitivity was 1 ng / ml . although the degree of precision of the elisa system in terms of intra - assay cv was between 4.0 and 10.0% , interassay cv was between 6.3 and 9.0% .
specificity was determined by human adiponectin , ctrp-1 , ctrp-2 , ctrp-3 , ctrp-6 , ctrp-7 , ctrp-9 , ctrp-10 , ctrp-11 , and ctrp-13 . no cross - reactivity was noted .
the same holds true for > 20 adipokines or metabolic hormones ( data not shown ) .
visceral and subcutaneous fat area was quantified by ct scan ( brilliance 64 ; philips medical systems , cleveland , oh ) .
visceral fat area ( vfa ) , total abdominal fat area , and subcutaneous fat area ( sfa ) were calculated from a 10-cm ct slice image between the fourth and fifth lumbar vertebrae that was obtained during suspended respiration .
ankle - brachial index and brachial - ankle pulse wave velocity ( bapwv ) were measured with a colin waveform analyzer ( model bp-203rpe ii ; nippon colin , komaki , japan ) .
data are mean  sd or median ( interquartile range [ 2575% ] ) . differences between groups were tested with an independent two - sample t test or mann - whitney u test for continuous variables , and differences in the distribution of categorical variables were tested with a  test .
spearman correlation test was performed to determine the relationships of ctrp-3 or ctrp-5 levels with other study variables .
multiple stepwise regression analyses with ctrp-3 or ctrp-5 as the dependent variable were performed to identify the independently associated factors in study participants .
differences between before and after the 3-month combined aerobic and resistance exercise program were tested with paired t test or wilcoxon signed rank test .
data were analyzed with sas version 9.2 ( sas institute , cary , nc ) statistical software .
the men were older and had higher body weight , bmi , waist circumference , blood pressure , and fasting plasma glucose , triglyceride , and liver transaminase enzyme levels than the women . furthermore , hscrp , ankle - brachial index , and bapwv values were significantly higher in men than in women .
clinical and laboratory characteristics of the study participants circulating concentrations of rbp4 were significantly higher ( p < 0.001 ) , whereas adiponectin levels were significantly lower ( p < 0.001 ) in men than in women ( table 1 ) .
women had significantly higher ctrp-3 levels than men ( median 429.9 [ interquartile range 344.6526.2 ] vs. 351.8 [ 279.2421.9 ] ng / ml , p < 0.001 ) .
ctrp-5 levels were also slightly higher in women than in men ( 50.4 [ 40.467.0 ] vs. 47.4 [ 36.459.8 ] ng / ml , p = 0.030 ) .
distributions of ctrp-3 ( a ) and ctrp-5 ( b ) in men and women . in a stepwise multiple regression analysis ,
log - transformed ctrp-3 levels were independently associated with age , sex , and triglyceride , ldl cholesterol , adiponectin , and rbp4 levels ( r = 0.182 ) ( table 2 ) .
in contrast , log - transformed ctrp-5 values were not significantly associated with other metabolic parameters ( data not shown ) . when obesity was defined as a bmi of 25 kg / m according to the criteria recommended by the korean society for the study of obesity ( 14 ) ,
ctrp-3 and ctrp-5 levels were not significantly different in participants with and without obesity ( p = 0.130 and 0.335 , respectively [ data not shown ] ) .
multiple linear stepwise regression analysis with ctrp-3 * value as a dependent variable after 3 months of a combined aerobic and resistance exercise program , body weight , bmi , waist circumference , systolic and diastolic blood pressure , and fasting plasma glucose , total cholesterol , ldl cholesterol , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , and homa - ir levels decreased significantly ( table 3 ) .
in particular , ctrp-3 levels decreased significantly ( median 444.3 [ interquartile range 373.8535.0 ] to 374.4 [ 297.2435.9 ] , p < 0.001 ) , whereas ctrp-5 levels increased slightly ( 34.1 [ 28.644.3 ] to 38.4 [ 29.855.1 ] , p = 0.048 ) after the combined exercise program ( supplementary fig .
clinical and laboratory parameters of study participants before and after a 3-month combined aerobic and resistance exercise program
the men were older and had higher body weight , bmi , waist circumference , blood pressure , and fasting plasma glucose , triglyceride , and liver transaminase enzyme levels than the women . furthermore , hscrp , ankle - brachial index , and bapwv values were significantly higher in men than in women .
circulating concentrations of rbp4 were significantly higher ( p < 0.001 ) , whereas adiponectin levels were significantly lower ( p < 0.001 ) in men than in women ( table 1 ) .
women had significantly higher ctrp-3 levels than men ( median 429.9 [ interquartile range 344.6526.2 ] vs. 351.8 [ 279.2421.9 ] ng / ml , p < 0.001 ) .
ctrp-5 levels were also slightly higher in women than in men ( 50.4 [ 40.467.0 ] vs. 47.4 [ 36.459.8 ] ng / ml , p = 0.030 ) .
distributions of ctrp-3 ( a ) and ctrp-5 ( b ) in men and women .
in a stepwise multiple regression analysis , log - transformed ctrp-3 levels were independently associated with age , sex , and triglyceride , ldl cholesterol , adiponectin , and rbp4 levels ( r = 0.182 ) ( table 2 ) .
in contrast , log - transformed ctrp-5 values were not significantly associated with other metabolic parameters ( data not shown ) . when obesity was defined as a bmi of 25 kg / m according to the criteria recommended by the korean society for the study of obesity ( 14 ) ,
ctrp-3 and ctrp-5 levels were not significantly different in participants with and without obesity ( p = 0.130 and 0.335 , respectively [ data not shown ] ) .
after 3 months of a combined aerobic and resistance exercise program , body weight , bmi , waist circumference , systolic and diastolic blood pressure , and fasting plasma glucose , total cholesterol , ldl cholesterol , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , and homa - ir levels decreased significantly ( table 3 ) .
in particular , ctrp-3 levels decreased significantly ( median 444.3 [ interquartile range 373.8535.0 ] to 374.4 [ 297.2435.9 ] , p < 0.001 ) , whereas ctrp-5 levels increased slightly ( 34.1 [ 28.644.3 ] to 38.4 [ 29.855.1 ] , p = 0.048 ) after the combined exercise program ( supplementary fig .
clinical and laboratory parameters of study participants before and after a 3-month combined aerobic and resistance exercise program
despite the multiple beneficial effects of adiponectin , the phenotype in adiponectin - null mice is relatively mild , even under diet- or genetically induced metabolic challenges . on the other hand , adiponectin gain - of - function studies reported definite improvements in the metabolic phenotypes .
therefore , this discrepancy suggests that compensatory mechanisms may exist that partially counteract the deficiency of adiponectin ( 15 ) .
the ctrp family of proteins comprises adiponectin paralogs that may allow cross talk between metabolic pathways and the innate immune system ( 16 ) .
recently , the ctrp family was proposed as a promising drug target in adipose tissue inflammation , insulin resistance , and type 2 diabetes ( 2 ) .
ctrp-3 ( also called cors-26 , cartducin , or cartonectin ) is synthesized and secreted by mesenteric adipose tissue in humans ( 17 ) .
( 18 ) found that ctrp-3 is positively regulated by insulin , whereas chronic lps exposure inhibits ctrp-3 expression .
ctrp-3 mrna expression is strongly induced during adipocyte differentiation , and knockdown of ctrp-3 in preadipocytes leads to dedifferentiation into a more proinflammatory and immature phenotype , which is characterized by upregulation of monocyte chemoattractant protein-1 ( mcp-1 ) release and reduction of lipid droplet size ( 3 ) .
ctrp-3 reduces interleukin-6 and tumor necrosis factor- secretion in lps - treated monocytic cells , and suppression of nuclear factorb ( nf-b ) signaling may explain the anti - inflammatory actions of ctrp-3 ( 19 ) .
( 20 ) reported that ctrp-3 stimulates the secretion of adiponectin and resistin in adipocytes , which is not caused by an induction of peroxisome proliferator 
ctrp-3 concentrations were positively associated with adiponectin levels , whereas they were negatively associated with rbp4 levels in multiple regression analysis .
further research should explore the interaction of adipokines , especially within the ctrp family of proteins .
ctrp-5 is expressed in adipocytes , with the highest expression being observed in the stromal vascular fraction ( 21 ) .
, the chromosomal localization of ctrp-5 falls within the linkage locus for obesity in pima indians , which suggests that ctrp5 might represent a candidate gene for obesity in humans ( 9 ) .
however , another study reported that ctrp-5 levels are not increased in the adipose tissue of ob / ob mice or in rosiglitazone - treated animals ( 21 ) .
furthermore , injection of mice with ctrp-5 has no effect on their glucose levels ( 21 ) . in the current study , which included a relatively large sample size , circulating ctrp-5 levels measured with a newly developed elisa
were not increased in obese human subjects compared with those of normal body weight ( p = 0.335 ) . on the other hand , depletion of mitochondrial dna results in insulin resistance as well as
moreover , phosphorylation of amp - activated protein kinase and acetyl - coa carboxylase is enhanced by ctrp-5 in human myocytes ( 8) . together , these results suggest that ctrp-5 is a metabolic mediator in the context of obesity and related insulin resistance ( 2 )
. however , the current study did not show a significant relationship between circulating ctrp-5 levels and insulin resistance index or other cardiometabolic risk factors .
moreover , circulating ctrp-5 concentrations were not associated with adiponectin or rbp4 levels in korean adults without diabetes . despite the structural similarities between adiponectin and the ctrp proteins , the metabolic effects of ctrp-5
in addition , although ctrp-5 has been identified as a marker of mitochondrial dysfunction in muscle , it is not yet clear whether muscle is a primary source of systemic ctrp-5 ( 2 ) .
the current study showed sex - specific differences in ctrp-3 and ctrp-5 levels , which are similar to trends found in adiponectin and several other adipokine levels .
for a given bmi , men have more visceral and hepatic adipose tissue , whereas women have more subcutaneous adipose tissue ( 22 ) .
this contrast may be attributable to different sex hormones and adipokines , which lead to more insulin - sensitive characteristics in women ( 22 ) .
previous studies have shown that adiponectin is higher in women than in men . even in children , adiponectin concentrations in girls are significantly greater than in boys ( 23 ) .
derived rbp mrna expression is sex specific and regulated by leptin . in the framingham heart study ,
rbp4 concentrations were higher in men and associated with insulin resistance and hscrp ( 25 ) .
the results of the current study are similar to previous reports in that adiponectin levels were higher and rbp4 levels were lower in women than in men .
wong et al . ( 21 ) reported that ctrp-5 protein levels were higher in the serum of female mice despite similar mrna expression of ctrp-5 in the adipose tissue of female and male mice .
in agreement with their findings , the current study showed higher circulating concentrations of ctrp-3 and ctrp-5 in women than in men .
these results suggest that the ctrp family of proteins might be one of the underlying factors explaining the difference in metabolic phenotypes between men and women . because adipokines influence insulin sensitivity and inflammation , regulation of adipokines may play a pivotal role in the etiology of insulin resistance , which is associated with obesity and type 2 diabetes .
exercise and weight reduction are common nonpharmacological interventions for the treatment of insulin resistance ( 26 )
. therefore , exercise may improve insulin resistance by regulating the circulating levels and/or functions of adipokines ( 26 ) .
( 27 ) , who reported that exercise reduces circulating rbp4 concentrations only in subjects who also experience improved insulin resistance . in previous studies , we observed a reduction in adipokine levels , such as visfatin and adipocyte fatty acid  binding proteins , which was associated with insulin resistance after a combined exercise program ( 28,29 ) . in the current study ,
we previously identified a paradoxical increase of ctrp-3 levels in insulin - resistant patients with type 2 diabetes , which may be a compensatory mechanism reminiscent of insulin or leptin resistance ( 6 ) .
recently , chalupova et al . ( 30 ) reported that ctrp-1 levels were significantly higher in subjects with metabolic syndrome than in healthy subjects . on the other hand ,
( 31 ) recently reported that ctrp-5 levels , as measured with immunoblotting , decreased significantly after exercise in 28 women .
there is no clear explanation for the discrepancy between the previous results and those of the current study .
however , the method ( immunoblotting vs. elisa ) , number of subjects ( 28 vs. 76 ) and type of exercise program ( aerobic vs. combined ) were different between the two studies , and the ctrp-5 levels during exercise showed only modest changes in both studies . in the current study ,
circulating adiponectin levels did not change significantly despite improved insulin sensitivity after 3 months of a combined exercise program , suggesting that exercise - induced improvements in insulin resistance develop independently of changes in circulating adiponectin levels ( 26 ) .
first , we used a cross - sectional design ; thus , it was not possible to draw conclusions about causality .
therefore , the results of the exercise study may not apply to men or other ethnic populations . in conclusion ,
similar to adiponectin , both circulating ctrp-3 and ctrp-5 concentrations showed sexual dimorphism , which is higher in women than in men . a 3-month combined aerobic and resistance exercise program significantly decreased ctrp-3 levels and modestly increased ctrp-5 levels , which was accompanied by improvements in cardiometabolic risk factors in obese korean women .
 objectiveto examine the effect of a combined exercise program on c1q / tumor necrosis factor - related protein ( ctrp ) 3 and ctrp-5 levels and novel adiponectin paralogs suggested to be links between metabolism and inflammation and to evaluate sex differences and association with cardiometabolic risk factors in humans with use of a newly developed elisa.research design and methodsthis cross - sectional study explored the implications of ctrp-3 and ctrp-5 on cardiometabolic parameters in 453 nondiabetic korean adults . 
 in addition , we evaluated the impact of a 3-month combined exercise program on ctrp-3 and ctrp-5 levels in 76 obese women . the exercise program consisted of 45 min of aerobic exercise at an intensity of 6075% of the age - predicted maximum heart rate ( 300 kcal / session ) and 
 20 min of resistance training ( 100 kcal / session ) five times per week.resultsboth ctrp-3 and ctrp-5 concentrations were significantly higher in women ( p < 0.001 ) than in men ( p = 0.030 ) . in a multiple stepwise regression analysis , 
 ctrp-3 levels were independently associated with age , sex , and triglyceride , ldl cholesterol , adiponectin , and retinol - binding protein 4 ( rbp4 ) levels ( r2 = 0.182 ) . 
 after 3 months of a combined exercise program , cardiometabolic risk factors , including components of metabolic syndrome , insulin resistance , and rbp4 levels , decreased significantly . 
 in particular , ctrp-3 levels decreased significantly ( median [ interquartile range ] 444.3 [ 373.8535.0 ] to 374.4 [ 297.2435.9 ] , p < 0.001 ) , whereas ctrp-5 levels were slightly increased ( 34.1 [ 28.644.3 ] to 38.4 [ 29.855.1 ] , p = 0.048).conclusionsa 3-month combined exercise program significantly decreased ctrp-3 levels and modestly increased ctrp-5 levels in obese korean women . 
RESEARCH DESIGN AND METHODS
Exercise protocol
Anthropometric and biochemical parameters
Development of human CTRP-5 ELISA
Measurement of visceral and subcutaneous fat area
Pulse wave velocity
Statistical analysis
RESULTS
Participant characteristics
Distribution of CTRP-3 and CTRP-5 according to sex
Determining factors for CTRP-3 and CTRP-5
Effects of an exercise program on physiological and biochemical parameters and adipokines levels
CONCLUSIONS