CHEMERIN IS AN INDEPENDENT MARKER OF THE METABOLIC SYNDROME IN A CAUCASIAN POPULATION – A PILOT STUDY

Aim: Chemerin is a novel adipokine that has been suggested to play an important role in the pathogenesis of the metabolic syndrome. The aim of our study was to evaluate serum chemerin as a marker of the metabolic syndrome and to assess its predictive accuracy in a Caucasian population. Methods: The study was designed as a cross-sectional study. Anthropometric measurements and serum analyses were done for Body Mass Index, waist circumference, chemerin, insulin, triacylglycerides, total cholesterol, HDLcholesterol, LDL-cholesterol, uric acid, and glucose in 55 non-obese healthy subjects and 181 subjects at risk for the metabolic syndrome. ROC curves were determined and the Chi-squared test was used to analyse the data. Results: Compared with healthy controls, subjects with suspected metabolic syndrome had signifi cantly higher chemerin serum levels (medians: 266.0 vs.192.5 μg/l; P < 0.01). After further chemerin adjustment, the diff erence between the subgroups persisted. Chemerin serum levels correlated with age (r = 0.23), serum glucose (r = 0.23), HDL-cholesterol (r = –0.19), triacylglycerides (r = 0.22), systolic and diastolic blood pressure (r = 0.40; r = 0.24) and the number of metabolic syndrome risk factors (r = 0.47). At a serum chemerin cut-off level of 240 μg/l, the presence of the metabolic syndrome was diagnosed with 75 % sensitivity and 67 % specifi city. Conclusion: In conclusion, serum chemerin levels are associated with the characteristics of the metabolic syndrome and could be an independent marker of this disorder in a Caucasian population.


INTRODUCTION
Adipose tissue is an active endocrine secretory organ which secretes a number of adipokines such as leptin, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), resistin, fatty acid binding protein-4 (A-FABP), adiponectin, chemokine ligand 2, PAI-1 (plasminogen activator inhibitor-1), adipsin, renin, angiotensinogen and angiotensin I and II .Adipocytes express receptors for some adipokines (e.g.TNF-α, adiponectin, leptin) that are reported to modulate lipid metabolism and adipogenesis directly by autocrine and paracrine mechanisms.Adipokines have important roles in the regulation of systemic lipid and glucose metabolism through systemic actions in the brain, liver and muscle.Adipokine secretion and blood level is mostly aff ected by the degree of adiposity, leading to the hypothesis that dysregulation of proinfl amatory and antiinfl amatory adipokines secretion in obesity may serve as a pathogenic link between obesity, insulin resistance and cardiovascular diseases 1,2 .
Chemerin is secreted as an 18kDa inactive pro-protein and undergoes extracellular serine protease cleavage of the C-terminal portion of the protein to generate the 16kDa active chemerin which is present in plasma and serum.It was originally reported to be present in circulation in plasma and serum, respectively, at 3.0 and 4.4 nM concentrations in humans, and 0.6 and 0.5 nM concentrations in mice.Active chemerin is abundant in ascitic fl uid from ovarian cancer patients (1.8-7.0 nM) and synovial fl uid of patients with arthritis (22 nM).However, tissues or cells responsible for chemerin secretion into these biological fl uids have not been established.
Recent fi ndings strongly suggest that white adipose tissue serves as both a primary source of chemerin secretion as well as a target for autocrine/paracrine chemerin signaling.A critical function of chemerin/ChemerinR is to regulate adipogenesis and metabolic homeostasis in adipocytes in mice and humans 2 .Expression of chemerin in white adipose tissue is much higher than in brown adipose tissue in lean subjects 2 .Chemerin and ChemerinR knockdown largely abrogate adipocyte diff erentiation.Thus, chemerin is essential in early diff erentiation processes and may contribute to or regulate critical early events in adipogenesis.Recent results also indicate that chemerin and ChemerinR could have an important biological role in the formation of white adipose tissue during normal development and in pathological states such obesity 2,5 .Chemerin downregulation during adipocyte maturation results subsequently in lower expression of perilipin, GLUT4 (insulin-regulated glucose transporter), adiponectin and leptin by mature adipocytes.This novel adipokine probably also modulates metabolic pathways in mature adipocytes.In agreement with this, the absence of chemerin expression results in a reduced basal and stimulated rate of lipolysis.On the other hand, nanomolar concentration of chemerin decreases intracellular cAMP (cyclic-adenosin monophosphate) leading to the assumption that chemerin might oppose the lipolytic action of catecholamines by reducing intracellular cAMP (2).In summary, chemerin has a regulatory role in adipogenesis and adipocyte metabolism, and infl uencing chemerin and ChemerinR signaling might to lead to novel therapeutic approaches in the treatment of obesity, diabetes mellitus type 2, and cardiovascular diseases 2 .
The aim of our study was to evaluate serum chemerin as a marker of metabolic syndrome by a new ELISA assay and to assess its predictive accuracy in Caucasian population.

Subjects
The study designed to be cross-sectional was approved by the ethics commission of the Sternberk Hospital, Czech Republic.It was conducted from January 2007 to November 2007 and included a total of 55 healthy individuals all of whom were non-obese, not on any medication and non-smokers), and 181 monitored individuals at high risk of the metabolic syndrome of which 122 were without the metabolic syndrome while 59 had it.Patients at risk for the metabolic syndrome were individuals who were monitored or treated for some indicator of the metabolic syndrome (hypertriacylglyceridemia, prediabetes, dyslipidemia, obesity).According to the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III), the metabolic syndrome is diagnosed when three or more of the following criteria are found in a subject: 1) waist circumference equal to or greater than 102 cm in men, or equal to or greater than 88 cm in women; 2) elevated triacylglycerides (equal to or greater than 1.7 mmol/l); 3) reduced high density cholesterol (HDL-cholesterol) (men -less than 1.0 mmol/l, women -less than 1.3 mmol/l); 4) elevated blood pressure (equal to or greater than 130/85 mm Hg or being treated for blood pressure lowering drugs); 5) Elevated fasting glucose (equal to or greater than 5.6 mmol/l) (ref. 6).Exclusion criteria of the study were as follows: acute or chronic systemic infl ammation (medical history, CRP> 10 mg/l, leukocytosis, clinical examination), diabetes mellitus treated with peroral antidiabetic drugs or insulin, liver diseases with transaminases levels above 5 times the normal upper limit, kidney diseases with creatinine levels above 130 μmol/l and/or proteinuria exceeding 0.5 g/l.

Sampling and laboratory determinations
Anthropometric, clinical, (height, weight, BMI, waist circumference, systolic and diastolic pressures) and laboratory analyses were performed.Fasting blood samples were drawn under aseptic conditions from vena cubiti after several minutes' rest in half-sitting position.Serum samples were separated by centrifugation at 4 °C at 3000 g for 20 min and subsequently used for analysis or frozen at -80 °C.

Statistical analysis
The data were analyzed using the statistics software package (MedCalc, Mariakerke, Belgium).Values of p < 0.05 were considered statistically significant.Spearman's correlation coeffi cients were used to establish the association between chemerin level and other parameters.Comparisons of chemerin serum values between risk subjects with and without the metabolic syndrome were made by either Student t test or Mann-Whitney test according to the distribution of the data.Frequency tables for the chi-squared test for chemerin values and the metabolic syndrome relationship and multiple regression for data adjustment to chemerin were done.Chemerin levels were also compared between risk groups with and without metabolic syndrome by covariant analysis using HDL-cholesterol, triacylglycerides, glucose, sex, age, BMI, WC and Quicki as covariates and adjusted using the linear regression model.The stepwise regression logistic model was also used.All data were expressed as medians or means ± standard deviations.Chemerin is an independent marker of the metabolic syndrome in a Caucasian population -a pilot study
The Chi-squared analysis confi rmed the relationship between serum chemerin and the diagnosis of the metabolic syndrome (Chi-squared 38.8; df 1; P < 0.01).
After adjustment for age and gender, chemerin levels were signifi cantly associated with metabolic syndrome related phenotypes (fasting glucose, fasting triacylglycerides, fasting HDL-cholesterol) but not with insulin sensitivity (Quicki) or adiposity (BMI, WC).After further adjustment for BMI, serum chemerin levels were still independently associated with metabolic syndrome related phenotypes, including blood pressure, serum glucose, HDL-cholesterol and triacylglycerides.The diff erence between both subgroups risk individuals (with or without metabolic syndrome) remained also after chemerin level adjustment for glucose, sex, HDL-cholesterol, age (P < 0.01) but not for triacylglycerides.These results clearly demonstrate that circulating chemerin levels are associated with key characteristics of the metabolic syndrome (Tab.3).In the stepwise logistic regression model, only chemerin, HDL-cholesterol and glucose were included for metabolic syndrome estimation.Percent of cases correctly classifi ed was 83.3 with an odds ratio 0.03 for HDL-cholesterol, 1.01 for glucose and 1.4 for chemerin.

DISCUSSION
The metabolic syndrome is a cluster of metabolic and cardiovascular disturbances, such as central obesity, hypertension, dyslipidemia and hyperglycemia.The source of chemerin protein in circulation is unclear at present.The highest chemerin expression is found in liver, adipose tissue and kidney.As chemerin mRNA expression is found to be increased in adipose tissue but not in the liver of obese compared to lean individuals, it is tempting to speculate that elevated chemerin levels found in plasma of obese humans may originate in adipose tissue 5 .As chemerin is a proinfl ammatory cytokine activating immune cells, it might play a role in the infl ammation of adipose tissue that occurs in obesity.Chemerin protein exists as a full length protein and a short form that is produced by removing 5 amino acids at the C-terminal end of chemerin protein by serine proteases, such as neutrophil elastase, cathepsin G and plasmin.The full length isoform of chemerin has signifi cantly lower biological activity than the proteolytically processed short form.It is unclear which form of chemerin is found in adipose tissue; however several known chemerin-activating proteases such is C1s, cathepsin G are present in adipose tissue.It has been hypothesized that chemerin may be proteolytically cleaved to the bioactive form in the adipose tissue of obese animals, whereas in lean animals, chemerin remains as the inactive full-length form 5 .Serum chemerin values were decreased in db/db mice 6 .In addition, chemerin potentiated insulin-stimulated glucose uptake in 3T3-L1 adipocytes concomitant with enhancing insulin signaling.ChemerinR is additionally expressed in macrophages suggesting that chemerin plays a potential role in regulating infl ammation by recruiting antigen-presenting cells.
The present study has shown that serum chemerin levels are associated with parameters of the metabolic syndrome and with hypertension regardless of the presence of metabolic syndrome components suggesting that chemerin may also be a novel regulator of blood pressure because of good correlations with both systolic and diastolic pressure.This hypothesis is supported by the fact that chemerin is highly expressed in the kidney, a key site of blood pressure regulation.Chemerin is structurally related to other circulating factors, including cathelicidins, cystatins, and kininogens; the proteolytic product of kininogen is the vasoactive peptide bradykinin.Similar results were recently presented in a large-scale epidemiological study from Mauritius 5 ; however, unlike that study, we found no relation between chemerin and adiposity.On the other hand, we detected a previously undocumented and signifi cant relation between serum glucose and chemerin.These diff erences might be due to the diff erent populations studied 5 .
In conclusion, our study indicates that circulating serum chemerin levels are associated with several key parameters of the metabolic syndrome in a Caucasian population.These fi ndings suggest that chemerin may play a role in the metabolic syndrome and might be an independent promising adipokine marker of metabolic syndrome.Further research is necessary to confi rm these fi ndings and to evaluate serum chemerin levels as a predictor of accelerated atherosclerosis or impaired glucose tolerance.

Table 3 .
Adjusted chemerin values in risk subjects with and without metabolic syndrome.Data presented as median (by data distribution).