recent evidence has shown that fat accumulation , especially in the abdominal cavity , causes dysregulation of adipokines , including increase in leptin , tumor necrosis factor- ( tnf- )  , interleukin-6 , and monocyte chemotactic protein-1 , and decrease in adiponectin , leading to the development of various metabolic disorders and atherosclerotic cardiovascular diseases [ 1 , 2 ] . adiponectin , a member of the c1q / tnf - related protein ( ctrp ) family , is one of the most extensively studied adipokines that possesses insulin - sensitizing , anti - inflammatory and antiatherogenic effects [ 35 ] . to date , 15 additional ctrp family members have been identified that are related to adiponectin in sequence and structural organization [ 6 , 7 ] . of all the ctrps , ctrp9 has the highest amino acid identity to adiponectin in its globular c1q domain [ 6 , 8 ] . ctrp9 is predominantly expressed in the adipose tissue and plays protective roles in diet - induced obesity , glucose intolerance , and insulin resistance in mice [ 8 , 9 ] . several basic studies have shown the beneficial effects of ctrp9 on the cardiovascular system [ 1014 ] . ctrp9 was shown to induce endothelium - dependent vasorelaxation , attenuate neointimal formation after vascular injury , and protect against cardiac injury [ 10 , 11 ] , and adverse cardiac remodeling   after acute infarction in mice . consistent with those experimental studies , serum ctrp9 levels were found to be inversely related to obesity , insulin resistance , and dyslipidemia in a community - based population   and in patients with coronary artery disease . in contrast , serum ctrp9 levels were positively associated with bmi in morbidly obese subjects requiring bariatric surgery   and with obesity , insulin resistance , and arterial stiffness in subjects with type 2 diabetes ( t2d ) . no study is currently available on the association between ctrp9 and morphological evidence of atherosclerosis in human subjects . moreover , the association of ctrp9 with atherosclerosis remains to be fully investigated because patients with renal dysfunction or chronic kidney disease ( ckd ) , who have a high risk of cardiovascular mortality , were not included in the preceding studies [ 15 , 16 , 18 ] . therefore , in this study , we measured the plasma levels of ctrp9 in subjects with t2d representing a wide range of renal function and investigated the clinical association of plasma ctrp9 level with the intima - media thickness ( imt ) of the carotid artery separately in subjects with ckd and in those without ckd . we consecutively enrolled 419 subjects with t2d ( 245 men and 174 women ) who were admitted to the diabetes center of the osaka city university hospital for the purpose of glycemic control , education , and/or evaluation of diabetic complications between january 2009 and july 2014 . subjects with type 1 diabetes and other types of diabetes were excluded from the present study . in our analyses , the estimated glomerular filtration rate ( egfr ) was calculated by using the japanese egfr equation , and the subjects were divided into the ckd ( egfr < 60  ml / min/1.73  m ) or the non - ckd ( egfr  60  ml / min/1.73  m ) group for analyses . this study was done in accordance with the declaration of helsinki ( 1975 , as revised in 2013 ) . the study protocol was approved by the ethics committee of our institution ( number 164 ) . all study participants provided written informed consent . blood pressure ( bp ) was determined by using the conventional cuff method with an automatic sphygmomanometer after the subjects had rested for at least 15  min . blood was drawn after an overnight fast , and biochemical parameters were analyzed by means of a standard laboratory method in the central clinical laboratory of the osaka city university hospital ( certification # 15 - 0240 by the japanese associations of medical technologists ) . glycated hemoglobin a1c ( hba1c ) was assessed as the national glycohemoglobin standardization program equivalent value ( % ) , which was expressed by adding 0.4 point to the hba1c ( jds ; % ) measured by standard laboratory methods and the previous japanese standard materials . immunoreactive insulin levels were measured for subjects not receiving insulin therapy ( n = 243 ) by electrochemiluminescence immunoassay ( cobas 8000(502/602 ) , roche diagnostics ) in the central clinical laboratory . homeostasis model assessment of insulin resistance ( homa - r ) was calculated according to the following formula : fasting insulin ( u / ml )  fasting glucose ( mg / dl)/405 [ 24 , 25 ] . plasma levels of ctrp9 ( ser877hu , uscn life science , houston , tx , usa ) and total adiponectin ( commodity code # 410614 , otsuka , tokyo , japan ) were measured by using an enzyme - linked immunosorbent assay following the manufacturer 's instructions . the minimum detectable levels of ctrp9 and adiponectin were 1.29  ng / ml and 23.4  pg / ml , respectively . the intra- and interassay coefficients of variation of ctrp9 were < 10% and < 12% , respectively , whereas those of adiponectin were < 10% and < 10% , respectively . ultrasonography of the common carotid artery ( cca ) was performed by using a phase - locked echo - tracking system , which was equipped with a high - resolution real - time 13  mhz linear scanner ( prosound ssd 6500 and f75 ; hitachi aloka medical , ltd . , the imt value was determined by using a measurement software ( intimascope ; media cross co. ltd , tokyo , japan ) , as described elsewhere . in brief , images were obtained 20  mm proximal to the origin of the bulb at the far wall of both ccas . the average value of 416 points in this region and the largest value , including plaque lesions , in the cca were measured separately . the mean - imt of the right and left cca ( mean - imt ) and the greatest imt among the left and right cca ( max - imt ) were used as markers of atherosclerotic changes . data are expressed as number ( % ) or median ( interquartile range ) as appropriate . for comparisons between the non - ckd and ckd groups , -test or wilcoxon rank - sum test multiple regression analyses were performed to explore the factors associated with carotid imt after adjustment for age , sex , body mass index ( bmi ) , systolic bp , egfr , hba1c level , triglyceride level , high - density lipoprotein ( hdl ) cholesterol level , low - density lipoprotein ( ldl ) cholesterol level , treatment with statins , treatment with angiotensin ii receptor blockers or angiotensin - converting enzyme inhibitors ( arbs / aceis ) , smoking status , ctrp9 level , and adiponectin level . a p value of < 0.05 was considered significant . statistical analyses were performed by using the jmp 10 software ( sas institute inc . , cary , nc , usa ) . the clinical characteristics of the total study population , as well as of the subjects with and without ckd , are shown in table 1 . the median age , duration of diabetes , and bmi of the subjects were 65 years , 11 years , and 25.0  kg / m , respectively . one hundred and sixty - one subjects ( 38.4% ) were categorized into the ckd group and the remaining 258 ( 61.6% ) were categorized into the non - ckd group . the median egfr was 42.7 and 76.7  ml / min/1.73  m for the ckd and the non - ckd group , respectively . as expected , subjects with ckd were older and had a longer duration of diabetes than those without ckd . the systolic bp and serum triglyceride levels were higher , and the hba1c , serum hdl - cholesterol , and ldl - cholesterol levels were lower in subjects with ckd than in those without ckd . the prevalence of subjects treated with oral hypoglycemic agents such as sulfonylureas , biguanides , and thiazolidinediones was lower , whereas that of subjects treated with insulin was higher , in the ckd group than in the non - ckd group . the ckd group had a higher prevalence of subjects treated with arbs / aceis and statins for hypertension and dyslipidemia , respectively , than the non - ckd group . the median plasma ctrp9 and adiponectin levels for the total population were 17.4  g / ml ( range , 0.0684.3  g / ml ) and 6.2  g / ml ( range , 0.8546.4  g / ml ) , respectively . of note , both plasma ctrp9 and adiponectin levels were inversely correlated with egfr ( figure 1 , supplementary tables  1 and  2 in supplementary material , available online at http://dx.doi.org/10.1155/2016/8624313 ) , and , accordingly , the plasma levels of ctrp9 and adiponectin were markedly higher in the ckd group than in the non - ckd group ( table 1 ) . no significant correlation between plasma ctrp9 and adiponectin levels was found in either the total population , the non - ckd group , or the ckd group ( supplementary table 1 ) . in univariate analyses , the plasma ctrp9 levels were positively correlated with age , systolic bp , serum creatinine , and triglycerides and negatively correlated with egfr , hba1c , and hdl - cholesterol ( supplementary table 1 ) . on the other hand , the plasma adiponectin levels were positively correlated with age , diabetes duration , systolic bp , serum creatinine , and hdl - cholesterol and negatively correlated with bmi , egfr , immunoreactive insulin , homa - r , and serum triglyceride levels ( supplementary table 2 ) . the median of max - imt and mean - imt was 1.08  mm ( range , 0.502.86 ) and 0.76  mm ( range , 0.341.57 ) , respectively . subjects with ckd had higher max - imt and mean - imt than those without ckd ( table 1 ) . we then examined the association of plasma ctrp9 levels with carotid imt by using univariate analyses in the non - ckd and the ckd groups , separately . in the non - ckd group , plasma ctrp9 levels were positively correlated with max - imt and mean - imt ( figures 2(a ) and 2(b ) ) . in contrast , neither max - imt nor mean - imt was significantly correlated with plasma ctrp9 levels in the ckd group ( figures 2(c ) and 2(d ) ) . on the other hand , plasma adiponectin levels were positively correlated with mean - imt (  = 0.147 , p = 0.019 ) , but not with max - imt (  = 0.036 , p = 0.563 ) , in the non - ckd group . neither max - imt (  = 0.039 , p = 0.627 ) nor mean - imt (  = 0.090 , p = 0.259 ) was significantly correlated with plasma adiponectin levels in the ckd group . finally , we performed multiple regression analyses to identify an independent association between plasma ctrp9 levels and carotid imt after adjusting for bmi , plasma adiponectin level , and other potential confounders including age , sex , systolic bp , egfr , hba1c , serum triglyceride level , hdl - cholesterol level , ldl - cholesterol level , smoking status , and presence of treatment with statins and arbs / aceis in the non - ckd or the ckd groups , separately . in the non - ckd group , plasma ctrp9 level was found to be an independent determinant of max - imt (  = 0.128 , p = 0.037 ) and mean - imt (  = 0.124 , p = 0.028 ) ( table 2 ) . notably , among variables other than ctrp9 , the independent determinants were only age for max - imt and only age and ldl - cholesterol level for mean - imt in the non - ckd group . on the other hand , no significant association was observed between plasma ctrp9 level and carotid imt in the ckd group ( table 2 ) . in the present study , we measured plasma ctrp9 levels in patients with t2d representing a wide range of renal functions and investigated the impact of plasma ctrp9 level on carotid imt separately in subjects with or without ckd . importantly , the association was independent of obesity , plasma adiponectin levels , and other traditional cardiovascular risk factors . the results also revealed that plasma ctrp9 levels were elevated in subjects with ckd compared with those without ckd ; however , plasma ctrp9 levels were not significantly associated with carotid imt in the ckd group . this study clearly demonstrated that plasma ctrp9 levels were independently and positively associated with carotid imt in diabetic subjects without ckd . previous experimental studies in mice consistently demonstrated the beneficial effects of ctrp9 on vascular endothelial function , vascular smooth muscle cell proliferation , and the profile of inflammatory cytokines in macrophages [ 28 , 29 ] . several studies have investigated the association of ctrp9 with cardiovascular risk factors in human subjects [ 15 , 16 , 18 ] , whereas only two reports have shown the association of ctrp9 with atherosclerosis   or cardiovascular disease . in a study in which 35% of subjects had diabetes , the ctrp9 level in serum and epicardial adipose tissue negatively predicted the presence of coronary artery disease . in contrast , a study exclusively of diabetic subjects   showed that serum ctrp9 levels were independently and positively associated with arterial stiffness . the present study showed a positive relationship between ctrp9 and carotid imt , suggesting that the impact of ctrp9 on atherosclerosis or cardiovascular disease differs depending on the characteristics of study population , at least with regard to diabetic status . unlike a prior study on subjects with t2d that evaluated subclinical atherosclerosis according to brachial - ankle pulse wave velocity , we were able to demonstrate in diabetic subjects a close relationship between plasma ctrp9 level and carotid imt , the most established surrogate marker for cardiovascular diseases . moreover , our subjects were older ( age , 65 versus 58 years ) and had poorer glycemic control ( hba1c , 8.6% versus 7.0% ) and lower renal function ( egfr , 76.7 versus 94  ml / min/1.73  m ) than those in a prior study , even in the non - ckd group . therefore , this study further suggests a significant impact of ctrp9 on atherosclerosis in patients with t2d who are exposed to a relatively high risk of cardiovascular diseases . importantly , the positive relationship between plasma ctrp9 levels and carotid imt was independent of obesity , plasma adiponectin levels , and other traditional cardiovascular risk factors in the non - ckd group . considering the inhibitory effect of ctrp9 on vascular smooth muscle cell growth and neointimal formation in mice , a detrimental impact of ctrp9 on carotid imt in this study could be explained by a compensatory response of ctrp9 to the conditions predisposing to the development of atherosclerosis in subjects with t2d . upregulation of ctrp9 expression in the adipose tissue was observed in 8-week - old ob / ob mice relative to age - matched controls . the protein expression of ctrp9 in cardiac tissue was also increased at 4 weeks in mice that received a high - fat diet and decreased thereafter . a prior study showing an independent and positive association of serum ctrp9 level with arterial stiffness in human subjects with t2d also indicated a role of ctrp9 as a compensatory factor in the pathogenesis of arterial stiffening . our research group previously reported that plasma adiponectin levels were inversely associated with carotid arterial stiffness in nondiabetic but not in diabetic subjects . no antiatherogenic effect of adiponectin with carotid imt taking these results together , a loss of antiatherogenic effect of adiponectin in diabetic condition could be one of the explanations for the compensatory response of ctrp9 to advanced atherosclerosis in patients with t2d . this study also suggests a possibility of a direct pathophysiological link between ctrp9 and atherogenesis in patients with t2d ; however , this needs to be confirmed with future studies . in this study , previous studies investigating ctrp9 levels in the circulation of human subjects excluded those with renal dysfunction [ 15 , 16 ] or targeted t2d subjects with normal renal function . this study is the first to demonstrate elevated plasma ctrp9 levels in human subjects with renal dysfunction compared with those without renal dysfunction . the plasma levels of adiponectin , which belongs to the c1q family and has high homology to ctrp9 , were also elevated in subjects with ckd and inversely correlated with egfr in the present study . the plasma levels of the other isoform of ctrp , ctrp3 , were also associated with egfr in human subjects including those with t2d . although the elevated adiponectin level in patients with ckd is reported to play a protective role against atherosclerosis and cardiovascular diseases [ 35 , 36 ] , it is currently unknown whether the elevated ctrp9 levels in subjects with ckd play a role or are merely due to reduced renal excretion . in the present study , the plasma ctrp9 levels were associated with systolic hypertension , renal dysfunction , and metabolic dysregulation ( e.g. , obesity , high triglyceride level , and low hdl - cholesterol level ) in the ckd group , whereas no parameter other than age and systolic bp was correlated with ctrp9 levels in the non - ckd group ( supplementary table 1 ) . these observations may indicate an association of ctrp9 with obesity - related metabolic dysregulation in diabetic subjects with ckd ; however , its functional significance needs to be confirmed in further studies . in contrast to diabetic subjects without ckd , those with ckd exhibited no significant association between plasma ctrp9 levels and carotid imt . it is well recognized that individuals with ckd are at a higher risk for cardiovascular mortality than those with preserved renal function . in patients with ckd , not only a clustering of several traditional risk factors , but also nontraditional risk factors such as anemia , hyperphosphatemia , hyperhomocysteinemia , and inflammation play a role in cardiovascular damage . indeed , in the multivariate analyses ( table 2 ) , the explanatory factors including traditional cardiovascular risk factors along with plasma ctrp9 and adiponectin levels did not significantly explain max - imt ( r = 0.088 , p = 0.515 ) or only minimally explained mean - imt ( r = 0.160 , p = 0.032 ) in the ckd group . therefore , it is possible to speculate that nontraditional factors related to ckd , although not evaluated in this study , attenuated the relationship between ctrp9 and carotid imt in subjects with ckd . the concentrations of circulating ctrp9 in this study population were found to be different from those in the prior studies [ 15 , 17 , 18 ] , even with the same assay kits . however , it needs to be mentioned that the ctrp9 levels were nearly one hundred times different among the prior studies ( mean 4.69  ng / ml , 115.3  ng / ml , and 415  ng / ml ) . moreover , ctrp9 concentrations obtained by the other assay kits were quite different between the assays ( 96  pg / ml   and 5.0  ng / ml ) . based on these facts first , we examined only ctrp9 based on its high similarity to adiponectin and the abundance in experimental evidence of its vascular effects [ 1014 ] . since ctrp1   and ctrp3 [ 34 , 40 ] were also indicated to be linked to atherosclerosis , the other members of the ctrp family need to be examined in the future . second , because this was a cross - sectional study , a causal relationship between ctrp9 and carotid atherosclerosis could not be confirmed . third , our subjects were receiving antihypertensive drugs and statins , which could have affected arterial thickness and its related risk factors . to minimize the effects of these drugs , we adjusted for the presence of these treatments in the multivariate analyses . fourth , no healthy controls were included in this study , and we could not confirm how diabetic status affects plasma ctrp9 level or its association with carotid imt . finally , because our subjects with t2d were hospitalized in a university hospital and were largely without adequate glycemic control , the current results can not be generalized to the entire population of patients with t2d . this study clearly shows that plasma ctrp9 levels are independently and positively associated with carotid imt in diabetic patients without ckd , but not in those with ckd . our data indicate an important link between ctrp9 and carotid arterial wall thickness , a powerful predictor of cardiovascular diseases , in patients with t2d . this study further proposes that plasma ctrp9 level is a potential biomarker of atherosclerosis in t2d patients without renal complications . further longitudinal studies are required to clarify whether plasma ctrp9 levels are predictive of the progression of atherosclerosis in patients with t2d who are at a high risk of cardiovascular diseases .
aim . 
 c1q / tumor necrosis factor - related protein-9 ( ctrp9 ) , a paralog of adiponectin , is expressed in adipose tissue . 
 ctrp9 exerts protective effects against obesity and atherosclerosis in rodents . 
 we investigated the association between plasma ctrp9 levels and atherosclerosis in patients with type 2 diabetes . 
 methods . 
 we included 419 patients with type 2 diabetes , 161 of whom had chronic kidney disease ( ckd ) . 
 fasting plasma ctrp9 and total adiponectin levels were measured with enzyme - linked immunosorbent assay . the intima - media thickness ( imt ) of the common carotid artery 
 was measured with ultrasonography . 
 results . 
 plasma ctrp9 levels were higher in the ckd group than in the non - ckd group . 
 plasma ctrp9 levels were positively correlated with carotid imt in the non - ckd group . 
 multivariate analyses revealed that plasma ctrp9 levels were positively associated with carotid imt in the non - ckd group , independent of age , sex , body mass index , adiponectin , and other cardiovascular risk factors . 
 however , plasma ctrp9 levels were not associated with carotid imt in the ckd group . conclusion . 
 plasma ctrp9 levels are associated with atherosclerosis in diabetic patients without ckd , independently of obesity , adiponectin , and traditional cardiovascular risk factors . 
 this study indicates a potential role of ctrp9 in atherosclerosis progression in human type 2 diabetes .