International Journal of Obesity (2009) 33, 1257–1264 & 2009 Macmillan Publishers Limited All rights reserved 0307-0565/09 $32.00 www.nature.com/ijo ORIGINAL ARTICLE Diminished upregulation of visceral adipose heme oxygenase-1 correlates with waist-to-hip ratio and insulin resistance

S Shakeri-Manesch1,3, M Zeyda2,3, J Huber2, B Ludvik2, G Prager1 and TM Stulnig2

1Department of Surgery, Medical University of Vienna, Vienna, Austria and 2Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna, Vienna, Austria

Objective: Insulin resistance in visceral obesity is substantially driven by adipose tissue inflammation, particularly by accumulating in obese adipose tissue. In contrast, adipose tissue macrophages express the scavenger receptor (CD163) and heme oxygenase-1 (: HMOX1) that together protect from oxidative stress. Our aim was to evaluate the expression of CD163 and HMOX1 in intra-abdominal visceral (omental) and subcutaneous adipose tissue as well as circulating soluble CD163 concentrations in human obesity and its association with adipose tissue inflammation, body fat distribution and insulin resistance. Methods: CD163, HMOX1 and CD68 mRNA expression in visceral and subcutaneous adipose tissue, serum concentration of soluble CD163 in morbidly obese patients (body mass index (BMI) 440 kg mÀ2), who underwent laparoscopic surgery for gastric banding (n ¼ 20), matched for age and sex to controls (BMIo30 kg mÀ2; n ¼ 20) was analyzed. Results: CD163 expression was highly upregulated in human adipose tissue and soluble CD163 serum concentration was elevated in obese vs lean subjects. HMOX1 was upregulated in adipose tissue by obesity as well and expressed predominantly in macrophages. Although CD163 expression strictly correlated with abundance, HMOX1 was additionally upregulated within macrophages. This upregulation was significantly lower in visceral compared with subcutaneous adipose tissue. Strikingly, relative visceral adipose tissue expression of HMOX1 negatively correlated with waist-to-hip ratio and the homeostasis model assessment of insulin resistance (both P ¼ 0.024). Conclusions: Visceral obesity is associated with defective upregulation of heme oxygenase-1 in visceral adipose tissue. A lack of this antioxidative and anti-inflammatory enzyme in visceral adipose tissue could contribute to the development of insulin resistance. International Journal of Obesity (2009) 33, 1257–1264; doi:10.1038/ijo.2009.160; published online 18 August 2009

Keywords: adipose tissue; inflammation; insulin resistance; macrophages; heme oxygenase

Introduction inflammatory response is located in adipose tissue.4 Within adipose tissue, inflammatory mediators are predominantly Obesity, particularly visceral obesity, which is reflected by an produced by non-adipocytes such as macrophages.5,6 Obe- elevated waist-to-hip ratio (WHR), is one of the major risk sity-associated adipose tissue inflammation is characterized factors for the development of insulin resistance, which is a by an increased abundance of adipose tissue macrophages in fundamental step towards mellitus.1 Obesity mice;6,7 several studies have confirmed a correlation between and insulin resistance are associated with a chronic low- body mass index (BMI) and adipose tissue macrophage grade inflammation that is determined by increased plasma numbers in humans particularly in the metabolically levels of inflammatory parameters in patients and different relevant visceral adipose tissue.8–11 Therefore, adipose tissue animal models of obesity.2,3 The main origin of this systemic macrophages are assumed to critically contribute to the pathogenesis of type 2 diabetes mellitus and the metabolic Correspondence: Professor TM Stulnig, Clinical Division of Endocrinology and syndrome by driving obesity-associated chronic inflamma- Metabolism, Department of Medicine III, Medical University Vienna, Wa¨hrin- tion. In contrast, adipose tissue macrophages may also have ger Gu¨rtel 18-20, Vienna 1090, Austria. cytoprotective anti-inflammatory properties, as identified by E-mail: [email protected] 3 the expression of several markers that determine an alter- These two authors contributed equally to this work. 12,13 Received 31 March 2009; revised 23 June 2009; accepted 4 July 2009; natively activated, anti-inflammatory macrophage type, 10,14 published online 18 August 2009 for instance, the hemoglobin scavenger receptor CD163. Adipose heme oxygenase-1 and insulin sensitivity S Shakeri-Manesch et al 1258 CD163 is exclusively expressed on and or partial colon resection (n ¼ 1). Adipose tissue samples were macrophages and mediates endocytosis of – taken from similar locations in all patients. Criteria for hemoglobin complexes,15 thereby preventing oxidative and exclusion were the presence of any infectious, inflammatory, nitrosative toxicity emerging upon red blood cell hemo- neoplastic or systemic disease, diabetes (excluded by fasting lysis.16 Moreover, CD163-mediated uptake of hemoglobin plasma glucose or the use of anti-diabetic drugs) or other into the macrophage fuels an anti-inflammatory response uncontrolled endocrine disease and the current use of driven by heme oxygenase-1 activity, leading to potent antibiotics, anti-inflammatory or anti-obesity drugs. The anti-inflammatory heme metabolites.17,18 A soluble form of study was approved by the ethics committee of the Medical CD163 (sCD163) of unknown function is found in serum University of Vienna. All subjects gave written informed and its concentration is elevated in , myeloid leukemia, consent before taking part in the study. Gaucher disease19 and atherosclerosis.20 Moreover, sCD163 serum concentration has been shown to increase with obesity in patients with chronic kidney disease.21 Metabolic parameter As CD163 is known to be highly expressed on adipose The HOMA-IR was calculated using fasting insulin and tissue macrophages, it may be speculated that the CD163/ glucose as described earlier.24 Characteristics of the study heme oxygenase-1 system is involved in obesity-driven population including serum C-reactive have been adipose tissue inflammation with potential consequences published elsewhere.25 on insulin sensitivity. Indeed, recent animal studies showed that the administration of heme oxygenase-inducers improve insulin sensitivity.22,23 In our study, we aimed at Analysis of gene expression investigating whether the expression of CD163 and heme Tissue specimens were washed in saline buffer, blood and oxygenase-1 (gene: HMOX1) is regulated by obesity in visible blood vessels were removed and tissues immediately subcutaneous and the metabolically more relevant visceral snap-frozen in liquid nitrogen. Total RNA was prepared by adipose tissue and whether the expression of these molecules disrupting adipose tissue in TRIzol reagent (Invitrogen, is related to adipose tissue inflammation, body fat distribu- Carlsbad, CA, USA) with a tissue homogenizer followed by tion and insulin resistance. We found that CD163 gene RNA isolation according the manufacturer’s instructions. expression is elevated in obese adipose tissue and strictly One mg of total RNA was treated with DNase I and reverse correlates with macrophage abundance in adipose tissue. transcribed into cDNA by Superscript II using random Adipose tissue HMOX1 expression was upregulated by hexamer priming (all Invitrogen). Quantitative real-time- obesity and exceeded macrophage abundance indicating PCR was carried out using gene-specific FAM-TAMRA-labeled additional upregulation within macrophages. Macrophage commercial Assays-on-Demand (Applied Biosystems, Foster HMOX1 upregulation by obesity was significantly lower in City, CA, USA) normalized to 18S VIC-TAMRA as endo- visceral compared with subcutaneous adipose tissue. Relative genous control (Applied Biosystems). Expression of specific visceral adipose expression of HMOX1 negatively correlated mRNAs in each sample was quantitated in duplicates on an

with WHR, indicating alterations in heme oxygenase-1 ABI PRISM 7000 Cycler (Applied Biosystems) using the DDCt regulation by the expansion of visceral adipose tissue that method. Values of the visceral adipose tissue of the control could abolish protection from oxidative stress. Moreover, group were generally taken as 100%. Relative visceral adipose visceral HMOX1 expression negatively correlated with tissue expression was calculated by dividing expression in insulin resistance as assessed by the homeostasis model visceral adipose tissue by the expression in subcutaneous assessment of insulin resistance (HOMA-IR). These data adipose tissue of the same subject. indicate that visceral heme oxygenase-1 expression is determined by body fat distribution and could be a factor preventing or attenuating obesity-induced insulin Immunoblotting resistance. Adipose tissue (300 mg) was homogenized and lysed on ice for 30 min in 0.5 ml tris-buffered saline, pH 7.4, containing 1% Triton X-100 and protease inhibitors. The tissue extract Methods was cleared from fat, nuclei and debris by centrifugation, and identical amounts of protein, as detected by BCA assay Patients (Pierce, Rockford, IL, USA), were separated by SDS-PAGE and Paired samples of visceral (omental) and subcutaneous blotted onto polyvinylidene fluoride membranes (Amersham, adipose tissue were obtained from 40 Caucasian men Little Chalfont, UK). Heme oxygenase was determined (n ¼ 12) and women (n ¼ 28). Morbidly obese patients (BMI using respective rabbit polyclonal antibody from BioVision 440 kg mÀ2) who underwent laparoscopic bariatric surgery (Mountain View, CA, USA) and b-acting using mouse mono- (n ¼ 20) were matched for age and sex with control subjects clonal antibody from Santa Cruz Biotech (Santa Cruz, (BMIo30 kg mÀ2) undergoing laparoscopic cholecystectomy CA, USA) followed by respective horseradish peroxidase- (n ¼ 15), fundoplicatio (n ¼ 3), resection of liver cysts (n ¼ 1) labelled secondary antibodies (Accurate, Westbury, NY, USA).

International Journal of Obesity Adipose heme oxygenase-1 and insulin sensitivity S Shakeri-Manesch et al 1259 Chemiluminescence was generated by BM chemilumines- cence substrate (Roche, Mannheim, Germany)

Adipose tissue cell fractionation Subcutaneous adipose tissue (n ¼ 5) was fractionated into cell populations as described previously.26 Briefly, visible vessels and connective tissue were carefully removed and tissue cut into pieces, washed in phosphate-buffered saline and digested for 1 h with 35 mgmlÀ1 liberase 3 (Roche) and 50 U mlÀ1 DNAse I (Sigma, St Louis, MI, USA). The digested tissue was passed through a 250 mM nylon mesh and centrifuged to obtain adipocytes (floating) and the stromal vascular cell fraction (pellet), and ‘matrix’.27 Following hemolysis, stromal vascular cells were subjected to magnetic-activated cell sorting of CD14 þ cells (Miltenyi Biotec, Bergisch Gladbach, Germany) to isolate adipose tissue macrophages. sCD163 Figure 1 Elevation of CD163 in obesity. Expression of CD163 (a)andCD68 A commercial ELISA (IQ Products BV, Groningen, The (b) mRNA was assessed in visceral (visc) and subcutaneous (sc) adipose tissue Netherlands) was used to quantitate sCD163 in serum of control (open bars) and obese (black bars) subjects (n ¼ 20 for each group). samples according to the manufacturer’s instructions. mRNA expression (mean±s.e.m.) is expressed in % of visceral adipose tissue from control subjects. (c) As an estimate of CD163 expression per macrophage, the ratio of CD163 and CD68 mRNA expression was calculated and is shown as mean±s.e.m. (d) Serum concentrations of sCD163 in control Statistical analysis (open bars) and obese (black bars). Significant results are indicated by All data are presented as mean±s.e.m. Repeated measures asterisks: *Po0.05; **Po0.01. analysis of variance was calculated in a factorial design by defining the factors BMI (obese/control) and location (visceral/subcutaneous adipose tissue). Post hoc analyses were indicating that CD163 expression in the adipose tissue is calculated by paired Student’s t-test and indicated in the directly linked to macrophage abundance without further figures. Pearson’s correlation was used for correlation regulation within macrophages. analysis. For comparison of groups determined by WHR Also sCD163 serum concentration was significantly and relative visceral HMOX1 expression, unpaired, two-tailed elevated in the obese (Figure 1d). However, sCD163 concen- Student’s t-test was used. Calculations were carried out using trations neither correlated with adipose tissue CD163 the statistical package SPSS version 14.0 (Statistical Package expression nor serum C-reactive protein within obese or for the Social Sciences, SPSS Inc., Chicago, IL, USA). control subjects (data not shown). A Po0.05 value was considered to indicate statistically signi- ficant differences. Regulation of heme oxygenase-1 expression in obesity In human adipose tissue, the gene for hemoglobin-metabo- Results lizing enzyme heme oxygenase-1 (HMOX1) was markedly upregulated by obesity (Figure 2a). HMOX1 gene expression Adipose tissue CD163 expression and serum sCD163 data were confirmed on the protein level by immunoblot concentration are elevated in obesity analyses (Figure 2b). Adipose tissue HMOX1 expression was The expression of the hemoglobin scavenger receptor gene essentially confined to CD14 þ cells consisting for the most CD163 was highly upregulated in human adipose tissue in part of macrophages (Figure 2d). The mean expression of obese compared with control subjects (Figure 1a). Upregula- HMOX1 in obese was about twice as high in subcutaneous tion by obesity was similar in intra-abdominal visceral compared with visceral adipose tissue, but this differences and subcutaneous adipose tissue. Similarly, expression of was not statistically significant because of large interindivi- the macrophage marker CD68 (Figure 1b) and macrophage dual variation (P ¼ 0.11; Figure 2a). The trend towards lower count25 was upregulated by obesity in both visceral and visceral adipose expression of heme oxygenase-1 in obesity subcutaneous adipose tissue to a comparable extent. CD163 was also apparent on the protein level (Figure 2b). As the expression strictly correlated with CD68 (r ¼ 0.952 and 0.920 expression of both HMOX1 (Figure 2d) and CD6826 is for obese visceral and subcutaneous adipose tissue, respec- confined to macrophages in adipose tissue and CD68 tively; Po0.001 for both). Accordingly, the ratio of CD163 accurately reflected the number of macrophages in adipose and CD68 expression in all groups was close to 1 (Figure 1c), tissue sections of the study population (Huber et al.25 and

International Journal of Obesity Adipose heme oxygenase-1 and insulin sensitivity S Shakeri-Manesch et al 1260

Figure 2 Regulation of heme oxygenase-1 expression in obesity. (a) Expression of HMOX1 mRNA in visceral (visc) and subcutaneous (sc) adipose tissue of control (open bars) and obese (black bars) subjects (n ¼ 20 for each group), expressed in % of visceral adipose tissue from control subjects. Significant results are indicated by asterisks: **Po0.01. (b) Immunnoblot analyses of adipose tissue lysates. Lysates of visceral (vi) and subcutaneous (sc) adipose tissue (n ¼ 12 for each group) were immunoblotted for heme oxygenase-1 (HO-1). Membranes were stripped and reprobed for b-actin. The Figure shows a representative blot of three donors from the obese (ob) and control (ctr) groups. (c) Subcutaneous adipose tissue was fractionated into matrix, adipocytes (AC), macrophage-depleted stromal vascular cells þ (SVC) and macrophages (CD14 ), as detailed in the Methods section. Figure 3 Waist-to-hip ratio (WHR) determines relative visceral Expression of HMOX1 was determined by quantitative real-time reverse HMOX1 expression. (a) WHR vs relative visceral adipose HMOX1 expression transcription-PCR and total adipose tissue (AT) was set to 100%. The diagram (HMOX1 /HMOX1 ) of female obese (r ¼À0. 579, P ¼ 0.024, n ¼ 16). ± visc sc shows mean s.e.m. of n ¼ 5. (d) As an estimate of heme oxygenase-1 (b–d) The total study population (b), as well as separately obese (c) and expression per macrophage, the ratio of HMOX1 and CD68 mRNA expression control (d), were divided into WHR low (WHRp0.9, female; WHRp1.0, male; in visceral (visc) and subcutaneous (sc) adipose tissue was calculated. n ¼ 23) and WHR high groups (WHR40.9, female; WHR41.0, male; n ¼ 17). *Po0.05; **Po0.01. Diagrams show relative visceral adipose HMOX1 expression of each donor expressed in mean±s.e.m. *Po0.05; ***Po0.001.

data not shown), the ratio of HMOX1 and CD68 was calculated as a measure of HMOX1 expression in adipose tissue macro- control subjects in our population but WHR was (obese phages. HMOX1/CD68 was significantly elevated by obesity in 0.945±0.034; control 0.893±0.023; P ¼ 0.014). Relative subcutaneous and visceral adipose tissue (Figure 2d). Of note, visceral HMOX1 expression in obese subjects negatively the increase in macrophage HMOX1 expression was signi- correlated with WHR (Figure 3a; r ¼À0. 579, P ¼ 0.024), ficantly less pronounced in visceral compared with subcuta- indicating that the relative amount of visceral fat could neous adipose tissue (P ¼ 0.020; Figure 2d). affect the relative expression of heme oxygenase-1. To further investigate the association of WHR and visceral heme oxygenase-1 expression, we divided the whole study High WHR is associated with low visceral adipose HMOX1 population into a WHR low (WHRp0.9, female; WHRp1.0, expression male; n ¼ 23) and a WHR high (WHR40.9, female; Visceral adipose tissue is more relevant for the development WHR41.0, male; n ¼ 17) group.29 As shown in Figure 3b, of insulin resistance than subcutaneous adipose tissue.28 The WHR high and low groups highly significantly differed in finding that the gene for cytoprotective and anti-inflamma- relative visceral HMOX1 expression. The significant differ- tory enzyme heme oxygenase-1 is less upregulated in visceral ence in relative visceral HMOX1 expression between subjects compared with subcutaneous adipose tissue lead us to the with high and low WHR was not only confined to obese question whether visceral obesity affects HMOX1 expression (Figure 3c) but also apparent in controls (Figure 3d) subjects. within visceral adipose tissue. Owing to high interindividual variation of adipose tissue HMOX1 expression (Figure 2a), we related visceral adipose tissue expression to that in sub- Low visceral HMOX1 expression is associated with insulin cutaneous adipose tissue taking subcutaneous adipose tissue resistance as an individual standard. Relative visceral HMOX1 expres- We calculated the relative visceral HMOX1 expression

sion was not significantly different between obese and (HMOX1visc/HMOX1sc) as a measure of the ability to

International Journal of Obesity Adipose heme oxygenase-1 and insulin sensitivity S Shakeri-Manesch et al 1261 A significant difference in HOMA-IR was also apparent when the obese group was divided according to the median

into high (HMOX1visc/HMOX1scX1.7)- and low (HMOX1visc/ HMOX1sc o1.7)-relative visceral HMOX1 expression sub- groups. As shown in Figure 4b, HOMA-IR significantly differed by about twofold between these subgroups of insulin-resistant obese subjects (P ¼ 0.0017), confirming low visceral HMOX1 expression to be associated with more severe obesity-induced insulin resistance (Figure 4b). When applying a cutoff level of 2 for the HOMA-IR to select a sufficient number of to some extent insulin-resistant subjects (n ¼ 8) in the control group, there was also a significant difference in HOMA-IR between subjects with high and low visceral HMOX1 expression (Figure 4c). These data indicate that low visceral adipose heme oxygenase-1 expression relates to insulin resistance independent of body weight. The tight relation of visceral adipose HMOX1expression and HOMA-IR was in trend (P ¼ 0.089), confirmed by a multi- variate regression model including WHR.

Discussion

Our data show that obesity is not only associated with the well-known inflammatory response in the adipose tissue, but also with a marked upregulation of the protective enzyme heme oxygenase-1. This upregulation could be a response to hypoxia that occurs in obese adipose tissue,31,32 because HMOX1 is a target gene of hypoxia-inducible transcription factors.33 Interestingly, in accordance with adipose tissue macrophages as shown here, also macrophages infiltrating hypoxic areas of human polycystic kidneys markedly express heme oxygenase-1.34 Importantly, the distribution of body fat, reflected by WHR, affects adipose tissue quality as assessed by HMOX1 Figure 4 Negative correlation of insulin resistance with relative visceral adipose heme oxygenase-1 gene expression. (a) HOMA-IR vs relative visceral expression. Visceral obesity indicated by high WHR is adipose HMOX1 expression (HMOX1visc/HMOX1sc) of obese subjects with an associated with a diminished upregulation of HMOX1 HOMA-IRX3(r ¼ 0.643, P ¼ 0.024). (b, c) The obese (b) and control (c) study expression levels in visceral adipose tissue, which is primarily groups were divided into high visceral (HMOX1visc high, HMOX1visc/ prone to be affected by obesity-mediated disturbances such HMOX1scX1.7) and low visceral (HMOX1visc low, HMOX1visc/HMOX1sc o1.7) HO-1 expression subgroups. HOMA-IR of 16 obese subjects with as adipocyte malfunction and death as well as inflammatory HOMA-IRX3(b) and 8 controls with HOMA-IRX2(c) are shown as responses,1,27,35 as compared with the more stable subcuta- mean±s.e.m. *Po0.05; **Po0.01. neous adipose tissue (Figure 3). Because of the negative correlation of relative visceral adipose HMOX1 expression upregulate HMOX1 in the metabolically relevant visceral with the HOMA-IR (Figure 4), heme oxygenase-1 is demon- compared with the subcutaneous adipose tissue, which is less strated as a presumable factor of visceral adipose tissue prone to obesity-induced alterations, thereby excluding quality that is most probably related to its antioxidative interindividual variations in adipose tissue gene expres- activity, albeit our study is limited to gene expression data. sion in general. To investigate whether relative visceral Of note, these associations exist not only in morbidly obese, HMOX1 expression relates to insulin resistance, we analyzed but also in non-obese individuals, indicating that accumula- correlations with HOMA-IR. Within obese insulin-resistant tion of visceral fat reduces its ability to dampen oxidative subjects (HOMA-IRX3 according to Cotrim et al.30) we found stress and interferes with insulin sensitivity already in the a significant correlation between HOMA-IR and relative lean individuals.36 The negative correlation of relative visceral HMOX1 expression (Figure 4a; r ¼ 0.643, P ¼ 0.024, visceral HMOX1 expression and HOMA-IR is evident only n ¼ 16), indicating that insulin resistance is linked to in insulin-resistant subjects. The fact that visceral HMOX1 low heme oxygenase-1 expression in visceral obesity. expression did not correlate with HOMA-IR in insulin-

International Journal of Obesity Adipose heme oxygenase-1 and insulin sensitivity S Shakeri-Manesch et al 1262 sensitive subjects does not necessarily mean that heme obesity (Figure 2d) is of particular interest and warrants oxygenase-1 is not important for insulin sensitivity in such further investigation. Such studies may also elucidate individuals, but suggests that more exact measures of insulin whether heme oxygenase-1 is regulate dependent or inde- sensitivity, particularly hyperinsulinemic euglycemic clamp pendent of CD163. testing, should be applied to estimate the putative role of Recent animal studies exploring the possibility to use heme visceral heme oxygenase-1. In conclusion, our data suggest oxygenase-1 to treat or prevent obesity-induced insulin that diminished visceral adipose heme oxygenase-1 expres- resistance show that administration of a heme oxygenase-1 sion may be a factor by which visceral obesity leads to inducers significantly improve insulin sensitivity and other insulin resistance. metabolic parameters in mice and rats.22,23 Although these The known cytoprotective, antioxidative and anti-inflam- improvements were associated with increased plasma adipo- matory properties of the CD163/heme oxygenase-1 nectin and decreased inflammatory and oxidative stress system16,18 together with the data on its adipose tissue markers, these data did not prove an involvement of adipose expression presented here, make low heme oxygenase-1 tissue inflammation and macrophages in the heme oxygenase- expression a putative powerful marker for detrimental 1-mediated effects. Moreover, treatment with heme oxy- adipose tissue quality. This notion is strongly confirmed by genase-1 inducer caused weight loss in one of these studies the negative correlation of relative visceral adipose HMOX1 that could underlie the metabolic effects.22 Nonetheless, the expression with the HOMA-IR (Figure 4). Moreover, bene- results of these studies strongly corroborate the conclusions ficial effects of a heme oxygenase-1 inducer/activator on of our study in humans that visceral adipose tissue heme adiponectin (an anti-inflammatory and insulin-sensitizing oxygenase-1 may be a regulator of insulin sensitivity and could adipocyte-specific cytokine37) plasma levels in rats and be used as a therapeutic target. in vitro adipogenesis have recently been described.38 Carbon Together with published findings the following model can monoxide, a main product of heme oxygenase-1-mediated be derived from our data: adipose tissue macrophages, which heme degradation, induces the expression of the transcrip- express the hemoglobin scavenger receptor (CD163), upre- tion factor PPARg in macrophages39 that drives anti- gulate protective heme oxygenase-1 upon obesity. However, inflammatory activation in macrophages and promotes accumulation of visceral fat interferes with this protective adipogenesis and adipocyte function.40,41 Thus, heme oxy- response by unknown mechanisms, leading to diminished genase-1 has a putative beneficial function in adipose tissue upregulation of visceral heme oxygenase-1 expression that through antioxidative, metabolic and anti-inflammatory results in a pro-inflammatory environment with increased mechanisms. oxidative stress that predisposes to insulin resistance. More Similar to adipose tissue CD163 expression, the serum detailed analyses including appropriate animal models and concentration of the soluble form of CD163 (sCD163) is in vitro analyses will reveal the validity of this model and elevated in obesity (Figure 1d), as previously shown in refine our knowledge on underlying mechanisms by which patients with chronic kidney disease.21 However, sCD163 visceral obesity and heme oxygenase-1 repression leads to concentrations did not directly correlate with adipose tissue metabolic deterioration. CD163 nor HMOX1 expression (data not shown). As no function has been described so far, sCD163 concentrations can currently be taken as an additional systemic inflamma- Conflict of interest tory marker in obese individuals, but our data fail to give hints of a relationship of sCD163 with the CD163/heme The authors declare no conflict of interest. oxygenase-1 system in adipose tissue macrophages. Clearly, further research is warranted to clarify the role of sCD163, particularly in obesity. Acknowledgements Macrophage CD163 expression that is known to be regulated by anti- as well as pro-inflammatory stimuli42 The research leading to these results has received funding was not apparently altered by obesity (Figure 1c). This from the European Community’s 7th Framework Programme finding is somewhat surprising, as factors inducing CD163 such as interleukin-6 and tumor necrosis factor-a are (FP7/2007–2013) under grant agreement no. 201608, from the Austrian Science Fund (project no. P18776-B11), and the elevated in the obese adipose tissue, but are in accordance Austrian National Bank (project no. 12735; all to TMS). with previous data on CD163 surface expression, which did not correlate with BMI in a small cross-sectional study.10 One explanation could be that CD163 expression is already very high in adipose tissue macrophages of lean individuals.10 References However, so far very little is known about molecules regulated by obesity in human in contrast to murine adipose 1 Despres JP, Lemieux I. Abdominal obesity and metabolic 13,43 syndrome. Nature 2006; 444: 881–887. tissue macrophages, and thus our results on HMOX1 2 Kahn SE, Zinman B, Haffner SM, O’Neill MC, Kravitz BG, Yu D indicating marked regulation in macrophages upon visceral et al. Obesity is a major determinant of the association of

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