The circulating levels of CTRP5 and the ratio of CTRP1 to CTRP5 plasma levels are significant predictors for cIMT value in patients with type 2 diabetes: A case-control study

Background: There is growing evidence that the C1qTNF-related protein (CTRP) family has a crucial role in the physiology and pathophysiology of metabolic disorders such as Type 2 Diabetes (T2D) and obesity. We sought to identify the association of CTRP1 and CTRP5 circulating levels with various obesity parameters such as visceral adipose tissue (VAT) thickness, visceral adiposity index (VAI), and with carotid intima-media thickness (cIMT) in patients with T2D and healthy subjects. Methods: This case-control study recruited subjects with T2D patients (n=42) as case group (all men) and without T2D (n=42) as controls (all men). cIMT and VAT thickness measurement was performed using an Accuvix XQ ultrasound. Circulating CTRP1 and CTRP5 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results: CTRP-1 and CTRP1/CTRP5 ratio were markedly higher in patients with T2D compared to controls (p < 0001 and p = 0004 respectively). Interestingly, binominal logistic regression revealed that a higher circulating level of CTRP1 was associated with the presence of T2D (odds ratio [OR]: 13203.554 [95% CI: 65.186-2674407.708]; P=.000). When considering the study population as a whole, CTRP1 circulating levels were correlated with WHR, VAT, and HOMA-IR. Also, we observed that the ratio of CTRP1 to CTRP5 plasma levels (β = 0.648, P=0.005) and CTRP5 circulating levels (β = 0.444, P=0.049) are significant predictors for cIMT value. Conclusions: Our results indicated that CTRP1 and CTRP5 concentrations were correlated with atherosclerosis in human subjects and these adipokines might have a causal role for cardiometabolic risk in type 2 diabetes disease


Background
Type 2 Diabetes (T2D) is one of the most common diseases worldwide affecting about 9% of the world population. (1) As a complex disease, it affects the function of many organs, which implies the possible connection it has with many other diseases (2)(3)(4).
Obesity and cardiovascular disease (CVD) which are in the context of this study, are two of the most well-known examples of these connections with confirmed links to diabetes (5)(6)(7).
Obesity is a leading cause of insulin resistance thus, it is considered as one of the major risk factors for diabetes. Moreover, it is generally accepted that CVD is the leading cause of mortality among T2D patients, which arise from several abnormalities in cellular metabolism and energy homeostasis. Since the connection among these conditions is becoming more and more evident through recent studies, identifying the possible molecules and their underlying mechanism seems to be important in this regard. (8) Adipose tissue plays a major role in regulating the overall metabolism of the body by using, secreting a wide range of adipocytes. Adiponectin and their paralogues, the family of C1q/TNF-related proteins (CTRPs), seems to be the crucial molecules in the cross-talk among metabolic disorders. (9) There is growing evidence that adiponectin levels are inversely associated with insulin resistance. Adiponectin acts as an anti-inflammatory agent in the adipose tissue and can improve insulin sensitivity in peripheral tissues. (10,11) More importantly, accumulating evidence points toward the association between hypoadiponectinemia and increased risk of cardiovascular problems. (12,13) The family of CTRPs has 15 protein members that play key roles in physiological and pathological metabolic conditions. (14,15) The alteration in circulating levels of CTRPs has been reported in various metabolic diseases such as T2D, obesity, CVD, fatty liver disease, and metabolic syndrome. (16)(17)(18)(19) CTRP1 is expressed in various tissues including adipose tissue, liver, muscles, kidneys, and heart. (14) Muscles are primary targets for CTRP1 as it activates AMPK and MAPK signaling pathways and promotes glucose uptake, ameliorates insulin resistance, and increases fat oxidation. (20) Studies have shown increased levels of CTRP1 in T2D, prediabetes, coronary artery disease, congestive heart failure, and atherosclerosis. (21,22) Although there is evidence on the protective role of CTRP1 in murine heart injuries, the exact role of CTRP1 in these conditions is still not fully understood and requires further studies. (23) CTRP5, another member of the CTRPs family also expressed in a wide range of tissues including adipose tissue, eye, testis, skeletal muscle, brain, spleen, uterus, and ciliary epithelium but like CTRP1, adipose tissue is considered as the main secretor. (21) CTRP5 increases glucose uptake via stimulating plasma membrane incorporation of the glucose transporter 4 (GLUT4) into the plasma membrane by a mechanism dependent on AMPK phosphorylation. Besides, Phosphorylation of acetyl-CoA carboxylase (ACC) is mediated by CTRP5 which results in fatty acid oxidation in rat myocytes. (14,24,25) According to recent studies, decreased levels of CTRP5 have been observed in metabolic syndrome, and T2D and increased levels were reported in coronary artery disease. Studies about the exact association between CTRP5 and metabolic disorders are rare with contradicting results, either suggesting a role against insulin resistance or a role promoting insulin resistance and atherosclerosis.
Given the importance of CVD as one of the most common causes of death in T2D patients as well as a close association among T2D, obesity, and an increased occurrence of CVD risk factors, it has been of great importance to detect the possible molecules which link between aforementioned conditions. Up to date, there is no study to evaluate CTRP1 and CTRP5 correlation with various obesity indices including body mass index (BMI), waist, hip, waist-to-hip ratio (WHR), visceral adipose tissue (VAT), and visceral adiposity index (VAI) in T2D patients compared to the control group. In this study, we intended to address the association of circulating CTRP1 and CTRP5 with obesity indices and carotid intima-media thickness (cIMT) in patients with T2D and control subjects.

Study population
A total of 84 subjects (all men) participated in this case-control study, including subjects with T2D (N=42) and subjects without T2D (N=42). All subjects were recruited from Shariati Hospital, Tehran, Iran. Outpatient clinic from March 2012 until November 2013. Informed written consent was obtained before the study, and the study was approved by the Ethics Committee of the Tehran University of Medical Sciences (TUMS). The participants were excluded if they had evidence of any chronic or acute systemic disease such as infectious disease, acute or chronic renal failure, malignancies, coronary artery diseases, and type 1 diabetes. It should be noted that none of the participants were current smoking.
To diagnosis, the T2DM, the basis of American Diabetes Association (ADA) criteria was used as fasting blood glucose (FBG) ≥126 mg/dl (7.0 mmol/l) or 2 hours plasma glucose ≥200 mg/dl (11.1 mmol/l) during an oral glucose tolerance test (OGTT) or random plasma glucose ≥200 mg/dl (11.1 mmol/l) (26). VAI was calculated as a novel sex-specific index, based on waist circumference (WC), BMI, triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), indirectly expressing visceral adipose function in all participants as described previously (27). We would like to stress that this study is reported in compliance with STROBE guidelines (Supplementary material).

Ultrasound methods
Ultrasound examinations for measurement of the cIMT and visceral adipose tissue thickness (VAT) were performed using an Accuvix XQ ultrasound unit (Medison, Seoul, Korea) equipped with a 3-7 MHZ curved-array and a 5-12 MHz linear-array transducer. The technique for measuring cIMT and VAT has been previously described (28,29). In brief, cIMT measured at its thickest point on the distal wall of the carotid arteries, along a 1.5-2 cm proximal to the carotid bulb. cIMT on the left and right sides was evaluated and mean values of both sides were determined as carotid IMT. Also, VAT (in millimeter) was measured as the distance between the anterior wall of the aorta and the internal face of the rectus abdominis muscle perpendicular to the aorta.

Anthropometric and clinical characterization
Anthropometric indices of all participants including age, weight, height, BMI, WC, hip, WHR, and blood pressure were examined. BMI was measured based on the ratio of weight in kg divided by height in m 2 to assess participants' obesity. WC using a flexible inch strip in the middle between the lowest rib and the iliac crest was calculated. Furthermore, the hip was measured at the maximum circumference of the buttocks. WHR based on the ratio of WC in centimeters divided by hip circumference in centimeters was measured. After a 15-minute rest in sitting position, systolic and diastolic blood pressures were measured by a manual sphygmomanometer. VAI, as a gender-specific mathematical index was calculated based on simple anthropometric [BMI and WC] and metabolic [TG and HDL Cholesterol (HDL)] parameters.

Plasma adiponectin measurement
Plasma levels of adiponectin were determined by using the ELISA Kit (Elabscience, Wuhan, China) according to the manufacturer's protocol. Intra-assay and inter-assay Coefficients of Variability (CV) were <10%.
2.6. Plasma CTRP5 and CTRP1 measurement CTRP1 concentration was measured by ELISA kit (Biovendor research and diagnostic products) with a minimum detectable concentration of 0.016 ng/ml, Intra assay Coefficients of Variability (CV) was 2.7% and Inter-assay CV was 8.5%. Plasma levels of CTRP5 were measured by immunoassay using the Cayman system kit according to the manufacturer's protocol. The inter-assay variability and intra-assay variabilities were 6.975 and 6.3 %, respectively.

Bias
Selection bias was addressed by closely matching cases to controls based on age. Moreover, all participants were men.

Statistical analysis
The sample size was calculated based on our previous studies. In detail, we estimated that considering alpha=0.05 and power=90%, a difference of 50% in the mean value of CTRP5 circulating levels between the two studied groups could be detected with a minimum of 30 subjects in each group.Here , we included 42 subjects in each groups.
Continuous variable with normal distribution is presented as mean ± standard deviation (SD) and variables with non-normal distribution are presented as median (interquartile ranges (IQR)). Descriptive analysis was applied and normality was tested for all quantitative variables using the Shapiro-Wilk test. For data with normal distribution, comparisons of anthropometric, biochemical, and laboratory parameters between two groups were done by the student's t-test and by the Mann-Whitney U test when we have non-normal distribution. A p-value <0.05 was applied to interpret all achieved data from analysis. All data analysis was performed using SPSS 20 (SPSS, Chicago, IL, USA).

Results
Anthropometric, clinical, and laboratory data of T2D patients and control subjects are shown in Table 1. All subjects were men and there is no statistical difference between two studied groups in terms of age (P =0.622). However, the T2D group had increased values of WC and WHR compared to controls (P=0.039 and P=0.012, respectively). However, other obesity parameters including hip, VAT, and VAI were not comparable between the two study groups. We should be noted that BMI was higher in T2D in comparison with the control group but did not reach our threshold of statistically significant difference.
As expected, patients with T2D had higher FBG concentration, insulin levels, and HOMA-IR in comparison with controls. The concentration of TG, HDL-C, LDL-C showed no significant difference between patients and controls.
All liver function-related tests including AST and ALT with expect to GGT showed higher levels in the T2D group compared to controls. Moreover, cIMT as a measurement of subclinical atherosclerosis was significantly increased in T2D patients compared to controls but did not reach our threshold of statistically significant difference (P=0.086).
The comparison of CTRP-5, CTRP-1, adiponectin circulating levels, and ratio of CTRP-1 to CTRP-5 between T2D patients and controls ( Figure 1) revealed that CTRP-1 and CTRP1/CTRP5 ratio were significantly higher in patients with T2D rather than in controls (P < 0001 and P = 0004 respectively). While, plasma levels of both CTRP5 and adiponectin with a borderline significant, was lower in T2D patients in comparison with controls (P =0.09 and P =0.094 respectively).
We also performed binomial logistic regression to investigate whether the ratio of CTRP1 to CTRP5 and plasma levels of CTRP1, CTRP5, and adiponectin might predict the presence of T2D (Table 2). When all afore-mentioned items were inserted, we found the one unit increase in logarithmic value of circulating level of CTRP1 was associated with the presence of T2D (odds ratio To identify independent predictors of CTRP1 circulating levels, we performed multivariate stepwise linear regression analysis with age, BMI, WC, hip, WHR, VAI, and VAT as independent variables. Our results showed that WHR (β = 0.273, P=0.014) is the only predictor for CTRP1 concentration in all participants.
Also, we performed multivariate stepwise linear regression analysis with cIMAT as a dependent variable and ratio of CTRP1 to CTRP5 and plasma levels of CTRP1, CTRP5, and adiponectin as independent variables. Our results showed that the ratio of CTRP1 to CTRP5 plasma levels (β = 0.648, P=0.005) and CTRP5 circulating levels (β = 0.444, P=0.049) are significant predictors for cIMT value.
The results of the correlation analysis of CTRP1/CTRP5 and circulating levels of CTRP1 and CTRP5 with anthropometric, and biochemical characteristics in all participants and T2D patients and controls are depicted in Table 3 and Table 4. We found a positive significant correlation between CTRP1 levels with WHR, VAT, and HOMA-IR in the whole population study. Moreover, there was a significant correlation between CTRP1 circulating levels with cIMT (P=0.062) (Fig 3a), VAI (P=0.093), and visceral fat (P=0.051) with a borderline significance. Moreover, CTRP5 circulating levels inversely correlated with the HOMA-IR index (p=0.008). Furthermore, the ratio of CTRP1 to CTRP5 was positively and significantly correlated with cIMT (P=0.020) (Fig 3b) and HOMA-IR (P=0.015) in all participants.

Discussion
A grown body of evidence highlight the crucial role of adiponectin and CTRP family in metabolic disorders (30,31), but few studies have ever attempted to associate the CTRP1, CTRP5, and adiponectin circulating levels with unfavorable obesity indices including VAI, VAT, HOMA-IR, and also carotid intima-media thickness in T2D patients. In our study, we have demonstrated that circulating levels of CTRP1 were higher in T2D patients compared to those in controls, in contrast to the reduced trend of adiponectin serum levels. Moreover, binominal logistic regression investigation revealed that elevated CTRP1 plasma levels were a direct indicator of T2D; suggesting the hypothesis that CTRP1 might be involved in the pathogenesis of T2D. Our results are consistent with observations reported by Bai B et al and Shanaki M et al (31,32). They found that plasma levels of CTRP1 increased in T2D and NAFLD patients relative to healthy participants. One possible explanation regarding the elevation of CTRP1 concentrations in T2D patients might be a self-protective or compensatory mechanism in response to abnormal glucose metabolism. Inconsistent with the above results, it was reported that CTRP1 levels did not significantly higher in diabetic subjects (33). The main reason for this discrepancy could be due to different samples as human and animal models (DIO or ob/ob mice) investigated. Also, in a previous study, it has been indicated that circulating CTRP1 in adiponectin-null mice were significantly enhanced relative to controls that confirms our finding concerning different serum pattern of CTRP1 and adiponectin (34). Also, CTRP1 may have specified overlapping roles as adiponectin. For instance, overexpression of recombinant CTRP1 could efficiently diminish serum levels of glucose in mice (34). Our data indicated that the differential pattern of CTRP1 and adiponectin in T2D condition might reflect an effective function in modulating glucose homeostasis; and the paradoxical elevation of CTRP1 circulating levels in T2DM subjects might exert a compensatory response to the abnormal glucose and lipid metabolism, which demands further clinical studies.
Interestingly, we found that CTRP1 markedly correlated with WHR and HOMA-IR and with VAI and VAT with a borderline significance. In line with our results, previous data reported that CTRP1 levels significantly increased in obese condition and associated with metabolic indices such as BMI and HOMA-IR (35)(36)(37)(38). Moreover, in line with current literature, Bai et al. observed higher circulating levels of CTRP1 were significantly correlated with hyperglycemia and HOMA-IR in T2D patients (39). However, contrary to this result, another study indicated that CTRP1 circulating levels significantly decreased in diet-induced obese mice relative to normal diet mice (36,40). Also, there is evidence that inhibition of CTRP1 impairs glucose homeostasis and insulin signaling which points into the possible contribution of CTRP1 in regulating energy metabolism and systemic insulin sensitivity (41,42). Moreover, it has been noted that CTRP1 likely correlated with sex hormones and may affect systemic metabolism in a sex-dependent manner (41). Based on discrepancy results of different patterns of CTRP1 in the context of obesity, future studies are needed to unravel underlying mechanisms in which CTRP1 regulates energy metabolism.
In the present study, a ROC curve analysis showed that circulating CTRP1 discriminated with high accuracy between T2D patients and controls. These findings point toward the contributory role of CTRP1 as a potential marker in patients with T2D. However, future study with a higher sample size is necessary to establish this concept.
There is also ample evidence about the possible role of CTRP1 in coronary artery disease and atherosclerosis (43)(44)(45). Besides, it has been shown that the circulating level of CTRP1 was associated with several diseased coronary arteries and the atherosclerotic extent index. Also, the close associations of CTRP1 with an unfavorable metabolic profile may have contributed to the significant linkage between CTRP1 and cardiovascular incidence risk. Here, we reported a positive correlation of CTRP1 with cIMT with a borderline significance. It has been noted that adiponectin (a paralogue of CTRP1) has potential anti-atherogenic properties and might be an independent factor correlated with atherosclerosis. So it is tempting to speculate that a high level of CTRP1 is independently associated with subclinical atherosclerosis and vascular injury.
Taken together, our results along with others suggest that measurement of circulating CTRP1 concentrations may be valuable for assessment of cardiovascular risk. However, future researches are required to elucidate the impact of CTRP1 on cardiovascular homeostasis.
Recently, CTRP5 has been found as a mediator of metabolic pathways involved in T2D, insulin resistance, and also an obesity-related cardiovascular risk (47). Nevertheless, the previous results are inconsistent. There is a report about the low levels of circulating CTRP5 in type 2 diabetes subjects, whereas another study argued that circulating levels of CTRP5 were significantly higher in obese and diabetic mice rather than lean group (47,48). An in vivo study by Lei et al revealed that CTRP5 circulating levels were not significantly changed in ob/ob mice (49). Besides, we previously showed that plasma CTRP5 levels were significantly lower in NAFLD and T2D patients in comparison with healthy subjects (50). In the present study, circulating CTRP5 concentrations were lower in T2D patients with a borderline significant level. As mentioned above, there is a discrepancy between animal model studies and human surveys that are might be due to the different functions of CTRP5 in humans and mice; just as resistin plays different roles in humans and mice (51). Another reason might be due to different genetic background that affects phenotype since type 2 diabetes in humans is a heterogeneous disease which was associated with environmental factors, whereas ob/ob and db/db mice are only caused by leptin deficiency (52). Also, it is noteworthy that we observed a negative correlation between CTRP5 and HOMA-IR, which suggests a link between CTRP5 and insulin resistance. It has been shown that inhibition of CTRP5 action may result in the alleviation of insulin resistance associated with obesity and diabetes (49). However, data are lacking on the correlation between CTRP5 and type 2 diabetes in humans and future clinical studies are demanded.
As an important finding, we also found that CTRP5 circulating levels and CTRP1/CTRP5 ratio might be two independent predictors for the cIMT index. It has been demonstrated that serum CTRP5 levels were significantly increased and are positively correlated with the extent and severity of atherosclerosis in patients with CAD (53). It seems that decreased levels of CTRP5 may be linked to cardiovascular complications in T2D patients. However, more research on this subject needs to be undertaken. It is tempting to speculate that the enhancement of CTRP1 levels along with reduced CTRP5 circulating levels was associated with an increase in the risk of T2D. Therefore, these adipokines can be considered as a possible metabolic mediator in the context of T2D and cardiovascular complications. It is also worth mentioning that the detailed mechanism associating with CTRP1 and CTRP5 alterations cannot be elucidated based on the current study, although we provided a novel finding regarding the association of CTRP1 and CTRP5 with T2D and cardiovascular risk. Several limitations of the study should be considered. The main limitation is the relatively small number of patients. In this regard, the low number of T2D patients precluded any definitive conclusions on the relationship between CTRP1, CTRP5, obesity indices, T2D, and cardiometabolic risk factors. In addition, we were able to recruit only men. Therefore, considering the sex-dependent manner of CTRP1 and CTRP5, it remains to be investigated the circulating levels of these adipokines in both genders.

Conclusions
In summary, we showed that circulating CTRP1 levels markedly elevated in T2D patients and significantly correlated with the cIMT index in all participants. Also, we revealed that CTRP5 may be a mediator of metabolic pathways that affects glucose and insulin metabolism in humans. We also noted that CTRP1 and CTRP5 concentrations were associated with atherosclerosis in human subjects and alteration in adipokines levels might be considered as an emerging cardiometabolic risk factor in patients with type 2 diabetes. However, more investigations are needed to confirm this concept.

Declarations Ethics approval and consent to participate
The procedures, used in the study, were approved by the Ethical Committee of the Tehran University of Medical Sciences (TUMS) and fully informed, written consent was obtained from the patients.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The study was funded by Tehran University of Medical Sciences,Tehran,Iran.

Acknowledgments
We are grateful to all of our patients who participated in this study. Data for plasma levels of CTRP1,CTRP5,and adiponectin were logarithmically transformed.CI indicates confidence interval; S.E., standard error of mean; OR, odds ratio. Tables 3 and 4 are only available as downloads in the supplementary files section. Figure 1 Plasma concentrations of adiponectin (a), CTRP1 (b), CTRP5 (c), and CTRP1/CTRP5 ratio (d) in non-T2D and T2D groups. The data are presented as median (Interquartile range). T2D: Type 2 diabetes. Independent Student's ttest on logarithmically transformed data was used to determine the differences of the adipokines between two groups.

Figure 3
Linear regression plot regarding the correlation of (a) CTRP1 circulating levels and (b) CTRP1/CTRP5 ratio with cIMT in the whole population. R coefficients and p-value were displayed. Data for CTRP1 circulating levels were logarithmically transformed.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Tables34.pdf