The Macrophage-Specific Serum Marker, Soluble CD163, Is Increased in Obesity and Reduced After Dietary-Induced Weight Loss

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1 The Macrophage-Specific Serum Marker, Soluble CD163, Is Increased in and Reduced After Dietary-Induced Weight Loss Karen Fjeldborg 1, Tore Christiansen 1, Marianne Bennetzen 1, Holger J. Møller 2, Steen B. Pedersen 1 and Bjørn Richelsen 1 Objective: Soluble CD163 (scd163) is a new macrophage-specific serum marker elevated in inflammatory conditions. scd163 is elevated in obesity and found to be a strong predictor of the development of type 2 diabetes. We investigated whether dietary intervention and moderate exercise was related to changes in scd163 and how scd163 is associated to insulin resistance in obesity. Design and Methods: Ninety-six obese subjects were enrolled: 62 followed a very low energy diet (VLED) program for 8 weeks followed by 3-4 weeks of weight stabilization, 20 followed a moderate exercise program for 12 weeks, and 14 were included without any intervention. Fasting blood samples and anthropometric measures were taken at baseline and after intervention. Thirty-six lean subjects were included in a control group. Results: scd163 was significantly higher in obese subjects ( mg/l) compared with lean ( mg/l, P < 0.001). Weight loss (11%) induced by VLED resulted in a reduction and partial normalization of scd163 to mg/l (P < 0.001). Exercise for 12 weeks had no effect on scd163. At baseline, scd163 was significantly correlated with BMI (r ¼ 0.46), waist circumference (r ¼ 0.40), insulin resistance measured by the homeostasis model assessment (HOMA-IR; r ¼ 0.41; all P < 0.001), and the leptin-to-adiponectin ratio (r ¼ 0.28, P < 0.05). In a multivariate linear regression analysis with various inflammatory markers, scd163 (b ¼ 0.25), adiponectin (b ¼ 0.24), and high sensitivity C-reactive protein (hs-crp; b ¼ 0.20) remained independently and significantly associated to HOMA-IR (all P < 0.05). After further adjustment for waist circumference, only scd163 was associated with HOMA-IR (P < 0.05). Conclusion: The macrophage-specific serum marker scd163 is increased in obesity and partially normalized by dietary-induced weight loss but not by moderate exercise. Furthermore, we confirm that scd163 is a good marker for obesity-related insulin resistance. (2013) 21, doi: /oby Introduction is associated with chronic low-grade inflammation, which may enhance the risk of developing insulin resistance, metabolic syndrome, and type 2 diabetes (1-3). We and others have shown that abdominal obesity in particular is associated with increased levels of inflammatory markers in the circulation and in the adipose tissue (4,5). Chronic low-grade inflammation is characterized by increased amounts of activated macrophages in the adipose tissue (6), which produce and release adipocytokines such as interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and monocyte chemoattractant protein-1 (MCP-1) (7,8). Soluble CD163 is one of the newer markers of macrophage activation (9). CD163 is a membrane-bound hemoglobin scavenger receptor involved in removing hemoglobin released from ruptured red blood cells. The receptor is primarily expressed on monocytes and macrophages. A soluble variant of CD163 (scd163) is present in the plasma and is elevated in pathological conditions with activation of the monocyte macrophage system (10 12). scd163 has antibacterial effects (13), and it is released from membrane-bound CD163 by the same enzymatic system that is involved in the release of TNF-a, suggesting that scd163 may be a surrogate marker for circulating TNF-a. However, scd163 is easier to measure in the 1 Department of Medicine and Endocrinology (MEA), Aarhus University Hospital, Aarhus, Denmark. Correspondence: Karen Fjeldborg (Karen.fjeldborg@ki.au.dk) 2 Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark Disclosure: The authors declare that there is no conflict of interest. Funding agencies: This study was supported by Aarhus University, Novo Nordic Foundation, and the Danish Agency for Strategic Research (TRAIN project). Received: 11 October 2012 Accepted: 26 December 2012 Published online 20 March doi: /oby VOLUME 21 NUMBER 12 DECEMBER

2 Soluble CD163 Is Partially Normalized After VLED Fjeldborg et al. circulation, since it has a much longer half-life than TNF-a (14). Recently, scd163 was found to be a strong predictor of the development of type 2 diabetes in a large Danish cohort (15). Other studies have unequivocally shown that scd163 is elevated in obese subjects compared to lean subjects (16-20). Furthermore, two recent studies have now shown that scd163 is positively associated with insulin resistance (measured by homeostasis model assessment, HOMA-IR) (18,19). Thus, scd163 may reflect the link between low-grade inflammation and metabolic abnormalities in the obese state. Besides scd163, other newer predictors of HOMA-IR have also been introduced recently. One of these is the leptin-to-adiponectin ratio (LAR), which has been more closely related to insulin resistance than leptin or adiponectin alone (21). The primary objective of the present study was to evaluate the regulation of circulating scd163 in relation to body weight and weight loss as well as to investigate whether scd163 is a useful marker for health complications in obesity, such as insulin resistance, compared to other inflammatory markers including the newer marker for insulin resistance LAR. Methods Subjects and the interventions The subjects in the present study were included from two different intervention studies in obese subjects and a case control study, comparing obese and lean subjects at baseline. The first intervention study consisted of a weight loss intervention with very low energy diet (VLED; DIO-1) which was compared with a lean control group (22). In the second study, obese subjects were randomized into one of the three groups investigating the effect of (1) weight loss induced by VLED (DIO-2), (2) exercise only (EXO), and (3) VLED in combination with exercise (DEX) (23). Together 96 obese subjects were included in the present study (47 males and 49 females). None of the subjects had type 2 diabetes or took medicine that could affect inflammation or adipose tissue metabolism. Inclusion and exclusions criteria are described earlier (22,23). Thus, 62 subjects (DIO-1 þ DIO-2 þ DEX) underwent 8 weeks with a VLED, followed by a 3-week (61 week) period of weight stabilization. The diet consisted of a liquid diet of kcal/day (VLED, Nupo Copenhagen) including 200 g of fruits and vegetables daily and weekly motivating sessions with a dietician. Twenty-one of the subjects on VLED followed at the same time an exercise program (DEX) with three sessions every week for min with estimated energy expenditure of kcal/session for 12 weeks as described previously (23). Twenty subjects underwent a program with only exercise training (EXO) as described for the DEX group but without VLED. The weight loss interventions with VLED were similar in study 1 (DIO-1) and study 2 (DIO-2), and, therefore, these groups were added together (DIO). Four subjects from the DIO group dropped out during the intervention period but were included in the baseline analyses. Thirty-six lean subjects were included in the control group with a mean BMI of kg/m 2. Anthropometric and clinical measures are shown in Table 1. All participants were asked not to engage in excessive physical exercise or alcohol intake the day before or in the morning of clinical investigations. Both interventions and the case control study were performed at the University of Aarhus, and the study designs were as mentioned identically for the DIO groups. The study protocol TABLE 1 Demographics of lean and obese subjects at baseline Lean Obese P-value N Age, years NS Sex (female) (n and %) 19 (52.8%) 49 (51%) NS BMI, kg/m ** Waist, cm ** Cholesterol, mmol/l ** HDL, mmol/l NS LDL, mmol/l ** Triglyceride, mmol/l ** Glucose, mmol/l ** Insulin, pmol/l ** HOMA-IR 1.9 ( ) 3.4 ( ) ** scd163, mg/l ** IL-6, pg/ml NS hs-crp, mg/l NS Adiponectin, lg/ml ** MCP-1, pg/ml ** Leptin, ng/ml ** LAR ** Baseline data are given for the lean and obese groups. Data are mean 6 SD or relative frequency (%). Comparison of lean and obese subjects at baseline by unpaired t-test or Wilcoxon Mann Whitney rank sum test where appropriated. LAR, leptin/adiponectin ratio; Waist, waist circumference. **P-value < NS, non-significant. was approved by the local ethics committee in the county of Aarhus and followed the principles in the Declaration of Helsinki. All subjects were recruited via advertisements in the local newspaper. They all gave written informed consent to participate in this study. Determination of scd163, hormones, and metabolites Fasting blood samples were drawn from all study participants to measure scd163, metabolic parameters, and inflammatory markers. Blood samples were taken at baseline and again at week in the intervention group. In a subgroup, blood samples were also taken at 8 weeks, after the weight reduction period and before the weight stabilization period. scd163 was quantified in serum samples using an in-house enzyme-linked immunosorbent assay (ELISA), essentially as previously described (24). Briefly, rabbit anti-cd163 was coated onto microtiter wells and plates transferred to a BEP-2000 ELISA analyzer (Dade Behring, Eschborn, Germany). Samples (diluted 1:101) were added in duplicates and incubated for 1.5 h at 37 C. Monoclonal anti-cd163 (GHI/61, 3 lg/ml) was incubated for 1 h at 37 C, and peroxidase-labeled antibody (goat anti-mouse immunoglobulins, DAKO P447, lg/ml; Dako, Glostrup, Denmark) was incubated for 1 h at 37 C. Tetramethylbenzidine substrate solution (Kem-En-Tec, Taastrup, Denmark, Cat. No. 4380) was used. Samples were measured in duplicates, and mean levels were used. The assay was calibrated using serum traceable to purified human CD163. The in-house assay correlates well 2438 VOLUME 21 NUMBER 12 DECEMBER

3 to commercial ELISA. The limit of detection (lowest calibrator) was 6.25 lg/l. Adiponectin was measured using a human-specific highsensitive ELISA method (b-bridge International, USA). High-sensitive CRP was measured with an hs-crp enzyme immunoassay (DRG, Germany). IL-6 was measured with a high sensitivity ELISA (R&D Systems, USA), and MCP-1 was measured with a human ELISA DuoSet (R&D systems). Leptin and insulin were measured with an enzyme immunoassay (Mediagnost, Germany, and DAKO, Cambrigdeshire, England). Glucose was analyzed at the local university department of clinical biochemistry. The HOMA-IR index was calculated using the formula: fasting insulin (lu/ml) fasting glucose (mmol/l)/22.5 (25,26). Statistics Descriptive statistics for anthropometric and metabolic risk markers is presented in mean 6 SD. Baseline unpaired data were analyzed with an unpaired t-test or a Wilcoxon Mann Whitney rank sum test for those variables, which were not normally distributed. The paired data, before and after intervention, was computed with a Wilcoxon signed rank test or a paired t-test where appropriated. Univariate association analysis was performed to compare scd163 with other parameters, and here we used Spearman s rank correlation. To adjust for age and sex, we made a partial Spearman s correlation. To analyze the associations between HOMA-IR and other variables, we used Pearson s correlation on a log scale. Multivariate linear regression was then performed to adjust for different metabolic and anthropometric parameters again on a log scale. The chosen significance level was a two-tailed P-value of <0.05. The statistical software packet SPSS (SPSS, Chicago, IL) was used for all calculations. Graphs were made in Sigma-Plot. Results scd163: lean versus obese subjects Characteristics of the lean and obese groups at baseline are given in Table 1. As expected, the obese subjects had significantly higher BMI ( kg/m 2 vs kg/m 2 ) and waist circumference than the lean subjects (P < 0.001). Furthermore, the level HOMA-IR was significantly elevated in the obese subjects (P < 0.001). Circulating level of scd163 was significantly higher in the obese subjects as compared with the lean subjects ( mg/l vs mg/l, P < 0,001; Table 1 and Figure 1). Of the adipocytokines, adiponectin was significantly reduced in the obese subjects, whereas MCP-1 and leptin were significantly elevated as compared to lean subjects (all P < 0.001). There was no difference between IL-6 and hs-crp in the two groups. The new suggested marker for insulin sensitivity, the LAR, was significantly elevated in the obese subjects compared with the lean (P < 0.001; Table 1). Age and sex distribution were comparable between lean and obese subjects (Table 1). Effects of weight loss and exercise on scd163 In the exercise group (EXO), the weight loss approximated 3.5% (Table 2), and the intervention significantly increased VO 2 max by to l/min (P < 0.01, data not shown). There was no effect on HOMA-IR, circulating scd163 ( to mg/l, P ¼ 0.68) or other inflammatory markers in the exercise group (Table 2). Since the weight loss and the changes in scd163 were FIGURE 1 Levels of scd163 in lean subjects, in obese subjects at baseline, and in obese subjects after weight loss. Thirty-six lean subjects with a mean BMI of kg/m 2 were compared with 96 obese individuals with a mean BMI of kg/m 2. Fifty-eight subjects from the obese group underwent a diet-induced weight loss program with VLED for 8 weeks followed by 3-4 weeks of weight stabilization. The weight loss after 12 weeks was approximately 11%. The lines represent mean values. **P < (lean versus obese). ## P < (before and after weight loss in the obese group treated with VLED). similar in the diet-induced weight loss group (DIO) and the dietinduced weight loss þ exercise group (DEX), these two groups were added together for the following analyses. In the DIO and DEX group, weight loss was induced by VLED for 8 weeks followed by weeks of weight stabilization. The weight loss was approximately 11% ( kg to kg, P < 0.001; Table 2), and the waist circumference was also reduced by 11% (Dwaist: cm, P < 0.001). The level of circulating scd163 decreased from mg/l to mg/l (P < 0.001) after the weight loss (Figure 1). In addition, HOMA-IR was reduced by 25% (DHOMA-IR: HOMA-IR units, P < 0.001). Among the inflammatory markers, adiponectin and MCP-1 were significantly changed (Dadiponectin: lg/ml, P < 0.001; DMCP-1: pg/ml, P < 0.001), but there was neither change in hs-crp (P ¼ 0.50) nor in the level of IL-6 (P ¼ 0.62). Leptin decreased significantly (Dleptin: ng/ml, P < 0.001), and the LAR was also significantly decreased after weight loss ( ng/lg to ng/lg, P < 0.001). After 8 weeks (at the end of the VLED treatment), the relative change in scd163 was positively associated with the relative changes in body weight (r ¼ 0.36, P < 0.01) and with changes in waist circumference (r ¼ 0.28, P < 0.05, data not shown), but, after the weight stabilization period for further weeks, these associations disappeared. There was no association between changes in scd163 or other inflammatory markers and changes in HOMA-IR during the weight loss intervention. The only variables that were significantly associated with changes in HOMA-IR were cholesterol (r ¼ 0.28) and LAR (r ¼ 0.31, both P < 0.05, Table 2). Relationship between scd163, BMI, and HOMA-IR at baseline All participants (lean and obese) are included in the following correlation analysis at baseline (n ¼ 132). In a univariate linear VOLUME 21 NUMBER 12 DECEMBER

4 Soluble CD163 Is Partially Normalized After VLED Fjeldborg et al. TABLE 2 Effects of exercise and weight loss induced by VLED EXO (n ¼ 20) DIO þ DEX (n ¼ 58) Baseline 12 weeks a Baseline 12 weeks a HOMA-IR (r) b Correlation with Weight ** ** 0.28* Waist ** ** 0.13 BMI ** ** 0.33* Cholesterol * ** 0.28* HDL LDL ** 0.20 Triglyceride ** 0.09 Glucose ** Insulin ** scd ** 0.06 IL hs-crp Adiponectin ** 0.04 MCP * ** 0.05 Leptin ** 0.25 LAR ** 0.31* Obese subjects (n ¼ 78) with a mean BMI of received three different weight loss interventions with (1) exercise alone (EXO), (2) VLED (DIO), and (3) the combination of both VLED þ exercise (DEX). The VLED was performed for 8 weeks followed by 3-4 weeks of weight stabilization. The exercise training was performed for 12 weeks. For the present analysis the DIO group and the DEX group are taken together because the changes concerning weight loss, scd163, etc., were similar between the two groups. Data are mean 6 SD or relative frequency (%). (a) Baseline versus end of the study (paired t-test or Wilcoxon Mann Whitney Rank Sum Test where appropriated). (b) Association between the absolute changes in HOMA-IR found during the weight loss in the DIO and DEX group (DDIO þ DEX) and the changes in the other measured variables (Spearman s correlation). r, correlation coefficient. The correlation coefficient is not given for insulin and glucose because of the relationship to HOMA-IR. *P-value < 0.05, **P-value < regression analysis, scd163 was found to be significantly positive associated with BMI (r ¼ 0.46, P < 0.001) and with waist circumference (r ¼ 0.40, P < 0.001; Table 3). Moreover, scd163 was significantly associated with HOMA-IR (r ¼ 0.41, P < 0.001; Table 3 and Figure 2). We did not find any significantly association between scd163 and other inflammatory markers but a positive association with leptin (r ¼ 0.25, P < 0.05) and LAR (r ¼ 0.28, P < 0.05; Table 3). The associations remained significant after adjusting for age and sex (data not shown). As shown in Table 3, scd163 was not affected by age, and the levels were similar between females ( mg/l) and males ( mg/l; P ¼ 0.90, data not shown). Other inflammatory markers (IL-6, MCP-1, hs-crp, adiponectin, and LAR) were also significantly associated with HOMA-IR (Table 3). Circulating scd163 was not significantly associated with blood lipids, whereas both adiponectin and LAR were significantly associated with high-density lipoprotein (HDL) cholesterol and triglycerides (Table 3). Relationship between HOMA-IR and scd163 and other inflammatory markers As shown in Table 3, all measured inflammatory markers were significantly associated with HOMA-IR at baseline. Thus, in order to sort out the important factors among the inflammatory factors related to HOMA-IR, a multivariate linear regression analysis was performed with all the inflammatory markers adjusted for sex and age. scd163 (b ¼ 0.25), adiponectin (b ¼ 0.24), and hs-crp (b ¼ 0.20) remained independently and significantly associated with HOMA-IR (all P < 0.05; Model 1, Table 4). When adjusting for waist circumference (surrogate index for abdominal fatness), only scd163 was associated with HOMA-IR (b ¼ 0.17, P < 0.05), whereas the association with adiponectin was not significant any longer (P ¼ 0.1; Model 2, Table 4). By adjusting for total fatness by BMI instead of waist circumference, the reverse results were found, where adiponectin was associated with HOMA- IR (r ¼ 0.20, P < 0.05) and scd163 was not (P ¼ 0.11; data not shown). When adding LAR to the inflammatory markers in the analysis, only scd163 (b ¼ 0.17, P < 0.05) and LAR (b ¼ 0.41, P < 0.05) were positively associated with HOMA-IR (Model 3, Table 4). Discussion This is the first study to show a significant reduction in the circulating macrophage marker scd163 upon diet-induced weight loss in obese individuals. Low-grade inflammation in obesity is closely related to the development of insulin resistance, type 2 diabetes, and other metabolic health complication (27,28) The background for low-grade inflammation is related to enhanced infiltration of activated macrophages in the adipose tissue in the obese state (6), and the biochemical characterization of this condition relies on markers in the serum 2440 VOLUME 21 NUMBER 12 DECEMBER

5 TABLE 3 Univariate association between scd163, adiponectin, LAR, and HOMA-IR and selected variables at baseline scd163 Adiponectin LAR HOMA-IR r r r r Age Waist 0.40** 0.44** 0.70** 0.56** Weight 0.45** 0.37** 0.66** 0.56** BMI 0.46** 0.22* 0.77** 0.57** Fasting glucose 0.30** 0.28* 0.30* 0.50** Insulin 0.38** 0.34** 0.57** 0.99** HOMA-IR 0.41** 0.34** 0.57** Cholesterol * HDL ** 0.26* 0.31* LDL * Triglyceride * 0.34* 0.31* IL ** 0.23* scd * 0.41** MCP * 0.19* Leptin 0.25* ** 0.46** Adiponectin ** 0.34** LAR 0.28* 0.4** 0.57** hs-crp ** 0.22* Obese and lean subjects at baseline (n ¼ 132). Statistically tests: Spearman s correlation, r, correlation coefficient. *P-value < 0.05, **P-value < released by activated macrophages (e.g., MCP-1, IL-6, and TNF-a) and adipocytes (e.g., adiponectin) (28). Presently, there is, however, no consensus in humans to which factor or combination of factors/ markers that can best describe the health complications related to low-grade inflammation. scd163 is a newer described marker of macrophage activation that can be measured in the circulation and which has been found both to be associated with obesity and to be a rather strong predictor of the development of type 2 diabetes (15). FIGURE 2 Correlation between HOMA-IR and scd163 at baseline. Data from lean and obese at baseline (n ¼ 132). Correlation coefficient (r) ¼ 0.41; P < (semi-scatter plot). TABLE 4 Multivariate linear regression for HOMA-IR and inflammatory markers Model 1 Model 2 Model 3 b P-value b P-value b P-value Adiponectin a 0.24 * 0.16 NS scd163 a 0.25 * 0.17 * 0.17 * IL-6 a 0.05 NS 0.04 NS 0.02 NS MCP-1 a 0.17 NS 0.08 NS 0.09 NS hs-crp a 0.20 * 0.10 NS 0.03 NS Sex 0.05 NS 0.05 NS 0.16 NS Age 0.02 NS 0.01 NS 0.01 NS Waist a 0.33 * 0.12 NS LAR a 0.41 * Obese and lean subjects at baseline (n ¼ 132). Model 1 includes inflammatory markers, adjusted for sex and age. Model 2 includes inflammatory markers, adjusted for sex, age, and waist circumference. Model 3 includes inflammatory markers and LAR (without adiponectin), adjusted for sex, age, and waist circumference. *P-value < 0.05; NS, non-significant. Dependent variable: HOMA-IR; b, regression coefficient. Log-transformed. In the present study, we examined a group of nondiabetic obese subjects to determinate the association between HOMA-IR and inflammatory markers, with focus on scd163 before and after weight loss. We found that scd163 was higher in obese compared to lean individuals and, moreover, that scd163 was positively correlated with both BMI and waist circumference. The level of scd163 was not affected by sex or age in this population. A recent study by Zanni et al. (18) reported similar finding in nondiabetic obese compared with lean subjects. Furthermore we found that weight loss (in the obese state) induced by VLED resulted in decreased level of scd163. The weight loss approximated 11%, and scd163 was significantly reduced nearly to the level seen in lean subjects. In the EXO group, performing moderate intensive exercise without dietary changes, no effect on neither HOMA-IR or on scd163 was found, even though this group lost about 3.5% of their body weight, indicating that exercise may have no effect on circulating scd163 or that exercise should be more intensive or more prolonged to affect scd163. One of the health complications to obesity is insulin resistance. In the present study, insulin resistance was measured by HOMA-IR. We found that scd163 was positively correlated with HOMA-IR at baseline with similar correlation coefficient as a well-known marker of insulin resistance, adiponectin (r ¼ 0.41 vs. r ¼ 0.34, respectively), indicating that scd163 may be a good marker for the insulin resistant state. The multivariate analysis emphasized this, since a positive association persisted between scd163 and HOMA-IR after adjusting for sex, age, and waist circumference, whereas the association to all the other inflammatory markers including adiponectin disappeared (Table 4). Interestingly, the changes in HOMA-IR during diet-induced weight loss did not correlate with the changes in scd163 or any of the other inflammatory markers after 12 weeks. Thus, scd163 seems to be closely related to HOMA-IR during baseline conditions, whereas this relationship disappears during the VOLUME 21 NUMBER 12 DECEMBER

6 Soluble CD163 Is Partially Normalized After VLED Fjeldborg et al. weight loss intervention, indicating that other factors than scd163 may be of importance for HOMA-IR during weight loss in obese subjects. The proposed new marker of insulin sensitivity LAR (21), which is the ratio between leptin (mainly a marker of total fatness) and adiponectin, was found to be strongly associated to HOMA-IR both at baseline (P < 0.001) and under the intervention (P < 0.05). LAR was also found to be significantly improved after the intervention with VLED. This finding is similar to a recent weight loss study by Oberhauser et al. (29). In a multivariate analysis with LAR, scd163 remained significantly associated with HOMA-IR but, however, to a lesser degree than LAR (model 3, Table 4). Circulating scd163 was not associated with any of the other measured inflammatory markers (Table 3) except of LAR (and leptin) which, however, may not be taken as a primary inflammatory marker. Additionally as a potential new marker, LAR requires two measurements (adiponectin and leptin) as compared to only one with scd163. In another recent study by Parkner et al. (19), scd163 was found to be a strong and independent predictor of insulin resistance in sex- and BMI-matched individuals with type 2 diabetes, impaired glucose tolerance, and nonimpaired glucose tolerance, which confirms our results. To further elucidate which factors/markers that are best suited to describe insulin resistance in the obese state LAR, scd163 and other markers should be related to the gold standard for investigating and quantifying insulin resistance (hyperinsulinemic euglycemic clamp). However, one should be aware of the fact that scd163 is affected by other factors than insulin sensitivity and metabolic conditions, since scd163 is also stimulated by more general macrophage activation in the body (e.g., infections and rheumatoid arthritis) (12). Among other health complications to obesity, and a part of the metabolic syndrome, is dyslipidemia (low HDL cholesterol and high triglyceride) (30). In the present study, scd163 was not associated with HDL cholesterol or with triglycerides (Table 3), which is in contrast to previous studies (15,19). This, however, may be due to a lack of power in the present study. Adiponectin (and LAR) was significantly correlated with both lipids (Table 3). Our data indicate that the hallmarks of the metabolic syndrome (insulin resistance, dyslipidemia, hypertension, etc.) may be the result of different pathogenetic mechanisms. It can be suggested that dyslipidemia may be primarily related to dysfunction of the adipocytes (adiponectin and leptin are the specific markers of adipocyte function), whereas insulin resistance may be related to both adipocytokines and specific macrophage activation, which can be determined by scd163. The strength of our study is the inclusion of a large number of subjects of both sexes and the strictly controlled exercise program and the VLED program, which allows us to study the individual effects of weight loss and exercise on CD163 and insulin sensitivity. This study had also some limitations. Moreover, the effect of more intensive exercise was not investigated. Insulin resistance was measured by the HOMA index, which, by itself, is a proxy for the gold standard (euglycemic clamp technique). Finally, we cannot tell from the present study if the decrease in scd163 is due to less numbers of macrophages in the adipose tissue or because of a phenotypic switch in the macrophages to a more anti-inflammatory profile. It is therefore of interest to examine the relationship between the gene expressions of different adipocytokines, scd163, and macrophage markers after weight loss in adipose tissue. In conclusion, we have confirmed that scd163 seems to be a good marker for obesity-related insulin resistance at least at baseline conditions. Furthermore, we now show for the first time that dietary intervention improves insulin sensitivity and at the same time lowers scd163. This study supports that scd163 in combination with other biomarkers may help to distinguish between healthy and unhealthy obese subjects.o Acknowledgments Pia Hornbek, Lenette Pedersen, and Kirsten Bank Petersen are acknowledged for their excellent technical assistance. VC 2013 The Society References 1. Stefan N, Kantartzis K, Machann J, et al. Identification and characterization of metabolically benign obesity in humans. Arch Intern Med 2008;168: Barbarroja N, Lopez-Pedrera R, Mayas MD, et al. The obese healthy paradox: Is inflammation the answer? Biochem J 2010;430: Dandona P, Aljada A, Bandyopadhyay A. Inflammation: The link between insulin resistance, obesity and diabetes. 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